MEMCAL    SCHOOL 
LUIBMAMY 


Panaina- Pacific  Intern 
iiXposition  Company. 


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CLINICAL    BACTERIOLOGY    AND    HEMATOLOGY 

FOR 

PRACTITIONERS 


CLINICAL  BACTERIOLOGY 
AND    Hy^MATOLOGY 

FOR 

PRACTITIONERS 


6^  v''  •      =^ 

WfD'ESTE  EMERY,  M.D.,  B.Sc.  Lond.    ' 

CLINICAL   PATHOLOGIST   TO   KING'S   COLLEGE   HOSPITAL   AND   PATHOLOGIST  TO   THE   CHILDREN'S 

HOSPITAL,    PADDINGTON   GREEN  ',    FORMERLY  ASSISTANT   BACTERIOLOGIST   TO   THE   ROYAL 

COLLEGES  OF  PHYSICIANS  AND  SURGEONS,  AND  SOMETIME  LECTURER  ON  PATHOLOGY 

AND   BACTERIOLOGY    IN   THE   UNIVERSITY  OF   BIRMINGHAM 


THIRD   EDITION 


PHILADELPHIA 
BLAKISTON'S   SON   &  CO.,  1012,  WALNUT  ST. 

1908 


PRINTED   IN   ENGLAND 


\  ^06 


PREFACE    TO    THIRD    EDITION 

In  making  such  alterations  as  seemed  called  for  in  the  prepara- 
tion of  this  edition,  an  attempt  has  been  made  to  avoid  as  far  as 
possible  any  further  expansion.  It  has  been  thought,  however* 
that  a  brief  account  of  the  methods  of  preparing  bacterial  vaccines 
should  be  inserted  :  not  that  the  practitioner  should  necessarily 
make  them  for  himself,  but  so  that  he  may  have  a  general  idea 
of  the  process.  Of  the  other  changes  made,  the  main  are  an 
account  of  making  cultures  of  the  blood  by  the  use  of  an  all- 
glass  exploring  syringe,  fuller  details  concerning  lumbar  puncture, 
the  diagnostic  value  of  this  operation  in  uraemia,  and  the  use  of  a 
Dewar's  flask  as  an  incubator. 

Some  of  the  coloured  plates  have  been  redrawn,  and  Figs.  31, 
34,  and  45  (for  which  I  have  to  thank  Mr.  R.  Gompertz)  added. 
My  thanks  are  also  due  to  Dr.  H.  B.  Day  for  Figs.  16,  17,  and 
26,  and  to  Messrs.  Down  Bros,  for  Fig.  30. 


W.  D'ESTE  EMERY. 


June,  1908. 


COMPLIMENTS 
OF 


kHsto&'s  Sob  Co.^ 


>i54()       r  '""    ^^ 


PREFACE   TO   SECOND    EDITION 

The  success  of  the  first  edition  of  this  book,  and  in  particular 
the  numerous  proofs  I  have  received  that  it  has  been  of  value  to 
those  for  whom  it  was  intended — that  is,  to  practitioners  who  are 
without  training  in  pathology,  and  who  want  a  simple  guide  to 
refer  to — has  encouraged  me  to  add  to  it  somewhat,  though 
without  altering  its  general  scope. 

The  additions  to  the  bacteriological  portion  are  mostly  concerned 
with  the  examination  of  materials  from  special  parts  of  the  body — 
mouth,  conjunctiva,  etc. — which  were  insufficiently  dealt  with  in 
the  first  edition,  and  are  treated  on  simple  lines  ;  in  most  cases 
the  diagnosis  by  microscopical  methods,  and  in  particular  by  the 
application  of  Gram's  stain,  is  given. 

The  haematological  portion  is  almost  all  new,  and  is  written  in 
response  to  numerous  requests  for  a  practical  guide  to  blood 
examinations,  especially  their  application  to  the  diagnosis  of 
disease.  I  have  attempted  to  deal  with  the  subject  on  the  same 
lines  as  the  bacteriological  portion — that  is,  to  explain  the  con- 
ditions under  which  a  certain  examination  is  indicated,  the  method 
employed  (in  full  detail),  the  deductions  to  be  drawn  from  the 
results  obtained,  and  the  cautions  necessary  in  interpreting  them. 
It  is  in  respect  of  these  interpretations  of  blood-counts  that  I 
think  such  a  guide  as  this  is  most  urgently  required,  as  I  find 
there  is  a  lamentable  ignorance  of  the  conditions  under  which 
blood-counts  are  required  and  the  methods  of  drawing  deductions 
from  them.  The  result  is  that  these  examinations  are  often  made 
unnecessarily  or  omitted  when  they  should  be  made,  that  the 
diagnosis  is  not  helped,  and  the  clinical  pathology  is  not  appre- 
ciated as  it  ought  to  be.     The  great  stumbling-block  has  been 


Vlll  PREFACE    TO    SECOND    EDITION 

that   the   blood-count   has   been  thought  to  give  the  diagnosis 
directly,  without  requiring  any  mental  exertion  on  the  part  of  the 
physician  or  surgeon.     It  does  so  in  some  cases,  but  in  most  it  is 
to  be  regarded  as  an  additional  fact  to  be  considered  in  making 
a  diagnosis — in  many  cases  the  chief  and  most  important  fact,  but 
one  that  must  always  be  considered  in  conjunction  with  those 
obtained  by  ordinary  clinical  methods.      The  results  of  a  blood 
examination  are  similar  to  those  of  any  other  physical  examina- 
tion ;  they  are  easy  to  obtain,  but  often  very  difficult  to  interpret, 
and,  just  as  in  the  diagnosis  of  the  diseases  of  the  chest  or  the 
nervous  system,  it  requires  much  experience  to  make  use  of  signs 
which  a  student  can  elucidate.     And,  as  in  the  case  of  the  other 
physical  signs,  the  blood-findings  are  sometimes  equivocal.     It  is 
about  as  reasonable  to  discredit  them  on  that  account  as  to  dis- 
credit percussion  of  the  chest  because  dulness  does  not  always 
indicate  pneumonia.     In  spite  of  these  difficulties  the  examination 
of  the  blood  is  one  that  is  more  likely,  in  many  cases,  to  give  a 
clue  to  what  is  actually  taking  place  than  is  any  other.    As  Cabot 
puts  it,  *'  The  blood  is  the  only  tissue  which  we  can  easily  examine 
during  the  life  of  the  patient.     Its  relations  to  all  other  tissues  are 
such  that  it  is  typical  of  them  all  in  a  way  that  no  other  tissue  is, 
acting  on  all  and  being  acted  on  by  all."     Here  I  have  attempted 
to  give  an  elementary  guide  to  some  of  the  more  important  and 
simple  changes,  and  for  further  reference  the  practitioners  are 
recommended  to  consult  one  of  the  largest  works  on  the  subject, 
such  as  those  of  Da  Costa,  Ewing,  or  Cabot,  all  of  which  are 
admirable  and  full  of  valuable  information. 

I  have  to  thank  Dr.  Whitfield  for  the  loan  of  the  very  admirable 
photographs  on  Plates  IV.  and  VI.,  and  for  revising  the  sections 
on  ringworm,  etc.,  and  Professor  Schaudinn  for  the  figures  of 
the  spirochaete  of  syphilis  shown  on  Plate  V. 

W.  D'ESTE  EMERY. 
March,  1906. 


PREFACE 

No  practitioner  who  wishes  to  do  his  best  for  his  patients  and 
to  promote  his  own  interests  can  afford  to  neglect  any  means  of 
cUnical  investigation  which  may  help  him  to  arrive  at  a  correct 
diagnosis,  and  offer  hints  as  to  prognosis  and  treatment.  Pre- 
eminent among  the  more  recent  methods  of  investigation  are  those 
which  are  applied  by  the  bacteriologist,  and  it  is  no  exaggeration 
to  say  that  in  many  of  the  infective  diseases  a  diagnosis  which  is 
made  without  a  bacterioscopic  examination  is  either  mere  guess- 
work or  can  only  be  made  so  late  that  the  patient  has  suffered 
unnecessarily  in  health  and  the  practitioner  in  prestige.  In  many 
cases,  however,  the  investigation  requires  a  considerable  amount 
of  technical  skill  and  access  to  a  well-equipped  laboratory.  The 
former  may,  perhaps,  be  possessed  by  the  rising  generation  (for 
bacteriology  is  now  an  integral  part  of  the  medical  curriculum), 
but  it  would  be  unfair  to  expect  every  medical  man  to  add  the 
latter  to  his  already  expensive  equipage.  But  in  many  cases  the 
diagnosis  can  be  arrived  at  by  very  simple  means — a  few  slides, 
cover-glasses,  and  stains,  a  good  microscope  (which  ought  to  be 
considered  as  essential  as  a  stethoscope),  and  a  very  moderate 
amount  of  technical  skill,  will  often  enable  the  practitioner  to 
arrive  at  a  correct  diagnosis  in  a  very  short  time.  This  little  book 
is  intended,  in  the  first  instance,  to  show  exactly  when  this  may  be 
done,  and  to  provide  clear,  succinct,  and  full  descriptions  of  simple 
methods  which  may  be  employed.  The  descriptions  of  the  opera- 
tions which  the  practitioner  can  carry  out  for  himself  are  mostly 
written  in  the  imperative  mood,  and  are  intended  to  be  referred  to 
constantly  and  carried  out,  step  by  step,  during  the  process.  They 
represent  the  instructions  which  would  be  given  by  a  teacher  when 


X  PREFACE 

watching  a  beginner  making  a  simple  bacteriological  examination 
for  the  first  time. 

It  cannot  be  too  strongly  urged  that  the  practitioner  should 
make  the  examination  for  himself  whenever  it  is  possible  for  him 
to  do  so,  and  if  not,  to  call  in  the  services  of  an  expert  pathologist. 
The  report  which  is  sent  from  a  public  laboratory  may  often  be  of 
very  considerable  value,  but  it  must  be  remembered  that  the 
bacteriologist  can  only  supply  facts,  and  the  inferences  which  may 
be  drawn  from  those  facts  will  largely  depend  upon  a  knowledge 
of  the  patient's  clinical  history  and  the  method  in  which  the 
material  was  obtained.  The  bacteriologist  is  too  often  in  the 
position  of  a  detective  who  has  to  unravel  a  mystery  from  obser- 
vations made  by  other  people,  and  has  no  opportunity  of  making 
investigations  for  himself.  A  bacteriological  examination  which 
is  made  by  one  person  and  interpreted  by  another,  or  which  is 
made  on  material  which  has  passed  through  more  hands  than  one, 
loses  much  of  its  value,  and  an  investigation  made  on  the  spot 
may  be  more  valuable  than  one  made  by  a  bacteriologist  of  far 
greater  experience  at  a  distance. 

The  methods  which  are  described  in  this  little  book  are  not  in 
all  cases  the  ideal  ones,  and  in  some  cases  they  are  somewhat 
different  from  those  which  are  generally  used,  but  they  are  simple 
and  efficient.  Of  course,  the  simple  examinations  which  are 
described  here  would  frequently  be  supplemented  by  more  com- 
plicated cultural  ones  by  a  trained  bacteriologist.  The  methods 
described  here  have  been  taught  in  the  post-graduate  classes 
which  were  initiated  some  two  years  ago  in  the  University  of 
Birmingham.  These  have  already  been  attended  by  about  a 
hundred  practitioners,  who  have  found  these  methods  of  great 
assistance  to  them  in  their  everyday  practice.  They  have  been 
selected  so  as  to  provide  examples  of  some  of  the  more  important 
operations  in  constant  use  in  the  bacteriological  laboratory.  The 
author  takes  this  opportunity  of  expressing  his  cordial  thanks  to 
Professor  Leith  for  his  kind  suggestions  as  to  the  general  scope  of 
the  book. 

No  apology  will  be  made  for  the  numerous  repetitions  which 
will  be  found  in  this  book.  They  are  essential  to  its  scope,  which 
is  to  give  clear  accounts  of  the  processes  with  as  little  reference  to 


PREFACE  XI 

Other  chapters  as  possible.     In  the  majority  of  cases  each  section 
is  complete  in  itself. 

These  instructions  are  followed  by  information  as  to  the  inter- 
pretation of  the  results  which  may  be  obtained,  and  this  informa- 
tion applies  equally  whether  the  medical  man  has  made  the 
examination  for  himself  or  has  obtained  it  ready-made  from  a 
public  laboratory.  It,  too,  commonly  happens  that  practitioners 
feel  themselves  aggrieved  because  they  get  a  negative  report  (as 
to  the  presence  or  absence  of  Widal's  reaction)  on  blood  taken 
during  the  first  few  days  of  an  illness  which  turns  out  to  be 
typhoid  fever,  or  are  inclined  to  discredit  bacteriological  examina- 
tions because  diphtheria  bacilli  are  found  in  throats  which  exhibit 
no  membrane  and  clear  up  in  a  few  days  without  serious  symptoms. 

In  the  second  place,  there  are  a  few  cases  in  which  the  investiga- 
tion had  better  be  made  in  a  public  laboratory.  In  these  the 
questions  of  what  to  send  and  how  to  send  it  are  fully  explained. 
This  is  a  most  important  point.  A  bacteriologist  is  not  a  magician 
who  is  able  to  weave  a  spell  if  he  has  a  small  portion  of  his 
victim's  anatomy  to  work  upon,  and  the  materials  must  be  taken 
in  the  proper  way  if  his  results  are  not  to  be  useless  or  even  mis- 
leading. This  is  well  seen  in  the  examination  of  the  blood  for 
bacteria.  In  many  cases  the  blood  is  drawn  in  such  a  manner 
that  it  must  necessarily  be  contaminated  from  the  skin  during  the 
operation,  and  is  transmitted  in  vaccine  tubes  which  were  almost 
certainly  not  sterile  before  being  filled.  Under  such  circumstances 
the  bacteriologist  will  probably  report  the  presence  of  streptococci 
or  staphylococci,  and  the  practitioner  who  does  not  understand  the 
fallacies  of  the  examination  may  be  led  to  make  a  diagnosis  which 
will  be  disastrous  to  his  own  reputation,  and  may  be  injurious  to 
the  patient. 

Lastly,  it  need  scarcely  be  said  that  this  is  not  intended  to  be 
a  substitute  for  any  one  of  the  numerous  excellent  works  on  the 
science  of  bacteriology  which  are  current  at  the  present  time. 
The  practitioner  is  strongly  recommended  to  supplement  the  very 
meagre  details  concerning  the  life-history  and  pathogenic  action 
of  the  bacteria  which  are  dealt  with  here  by  a  study  of  one 
of  these  text-books.  Muir  and  Ritchie's  admirable  "  Manual 
of    Bacteriology,"    Crookshank's    "  Bacteriology    and    Infective 


XU  PREFACE 

Diseases,"  Hewlett's  "  Bacteriology,"  Klein's  "  Micro-organisms 
and  Disease,"  McFarland's  '*  Text-book  upon  the  Pathogenic 
Bacteria,"  or  Curtis's  "  Essentials  of  Practical  Bacteriology," 
are  all  suitable  for  this  purpose,  and  a  perusal  of  any  one  of  them 
will  be  both  pleasurable  and  profitable  to  every  medical  man. 

Several  illustrations  have  been  borrowed  from  sources  mentioned 
in  the  text,  and  for  the  loan  of  these  the  author  wishes  to  express 
his  best  thanks  to  the  respective  authors  and  publishers.  His 
best  thanks  are  also  due  to  Messrs.  Baird  and  Tatlock,  Swift  and 
Son,  Zeiss,  Leitz,  Hawksley,  and  Hearson,  for  kindly  providing 
illustrations  of  apparatus  made  by  them. 


CONTENTS 

PART   I 
BACTERIOLOGY 

Section  I 
APPARATUS  AND  PROCESSES 

PAGE 

THE   BACTERIOLOGICAL   MICROSCOPE       -                 -  -  -            I 

STERILIZATION    OF    APPARATUS,    ETC.       -                  -  "  "            5 

PREPARATION    OF    CULTURE    MEDIA          -                  -  "9 

INOCULATION    OF    CULTURE    MEDIA           -                  -  -  -          l6 

INCUBATION    OF    CULTURES           -                  -                  -  "  -          l8 

METHOD    OF    EXAMINING    CULTURES         -                  "  "  -         22 

gram's    METHOD   OF    STAINING-                 -                 "  "  -24 

EXAMINATION    OF    FILMS — USE   OF    MICROSCOPE  -  -27 
STAINS  --------         28 

CLEANING   SLIDES    AND    COVER-GLASSES                  -  -  "         3^ 

PIPETTES               -                 -                 -                 -                 -  "  -32 

Section  II 
DIAGNOSIS  OF  CERTAIN  DISEASES 

DIPHTHERIA        -                 -                 -                 -                 "  "  "37 

TETANUS               -                 -                 -                 -                 -  -  -         45 

THE   PNEUMOCOCCUS,    PNEUMONIA,    ETC.                -  -  "49 

INFLUENZA           -                 -                 -                 -                 "  "  "52 

ANTHRAX              -                  -                  -                  -                  -  -  -         54 

TUBERCLE             -                  -                  -                  -                  "  "  "59 

LEPROSY                 -                  -                  -                  -                 -  '  -         ^5 

ACTINOMYCOSIS,    OR   STREPTOTHRICOSIS                 -  -  '         ^5 


XIV  CONTENTS 

PAGE 

GLANDERS            -                 -                 -                 -                                   -                 -  68 

TYPHOID    FEVER                 -                 -                                  -                 -                 -  69 

GONORRHCEA       -                 -                 -                 -                 -                 -                 -  84 

SYPHILIS                -                 -                 -                                  -                 -                 -  89 

CHOLERA              -                 -                 -                 -                 -                 -                 -  91 

PLAGUE                  -                 -                 -                 -                                  -                 -  93 

SOFT   SORE          ...---.  94 

RINGWORM           -                 -                 -                 -                 -                 -                 -  95 

OTHER    SKIN    DISEASES  ------  lOO 


Section  III 

COLLECTION   AND   EXAMINATION   OF   CERTAIN  MORBID 

MATERIALS 

THE   COLLECTION   AND    EXAMINATION    OF   PUS   -                 -  -  I02 

THE     BACTERIOLOGICAL     EXAMINATION     OF     THE     MOUTH  AND 

FAUCES              -                  -                                    -                                    -  -  105 

THE      BACTERIOLOGICAL       EXAMINATION      OF     THE      NOSE  AND 

ACCESSORY   CAVITIES                    -                  -                  -                  -  -  IIO 

THE   CONJUNCTIVA           -                 -                  -                  -                  -  -  III 

THE   SPUTUM      -                  -                 '                  -                  -                 -  -  113 

THE   GASTRIC    CONTENTS   AND   VOMIT     -                 -                 -  "US 

THE   URINE         -                 -                 -                 -                 -                 -  -  118 

THE   COLLECTION   OF    FLUIDS    FROM    SEROUS    CAVITIES    -  -  121 

THE   PLEURA      -                 -                 -                 -                 -                 •  -  1 23 

FLUIDS    FROM   JOINTS     -                 -                 -                 -                 -  -  1 26 

LUMBAR    PUNCTURE         -                 -                 -                 -                 -  -  1 28 

THE   BACTERIOLOGICAL   EXAMINATION   OF   THE    BLOOD  -  -  1 38 

EXAMINATION    FOR    BACTERIA    IN    FILMS                 -                 -  -  I41 

MALARIA  -.-....  142 

EXAMINATION    BY   CULTURAL   METHODS                  -                 -  -143 

ESTIMATION   OF   THE   OPSONIC    POWER    OF   THE   BLOOD  -  .  -  148 

COLLECTION   OF   MATERIAL   AT    POST-MORTEM    EXAMINATIONS    -  158 

SECTION-CUTTING               -                 -                 -                 -                 -  "159 

FIXING   MATERIAL   FOR   CUTTING               ...  -  161 

SECTION-CUTTING    BY   THE    FREEZING    METHOD  -                 -  "  163 

STAINING   AND    MOUNTING    FROZEN    SECTIONS     -                 -  '   -  165 


CONTENTS  XV 

PAGE 

THE   PARAFFIN    PROCESS  -  -  -  -  -  1 66 

DEHYDRATION        -  -  -  "  "  -  I 66 

CLARIFICATION      -  -  -  -  -  -  167 

INFILTRATION    WITH    PARAFFIN      -  -  -  -  1 67 

CASTING   THE   BLOCKS         -----  16^^ 

CUTTING    THE    SECTIONS    -  -  -  -  -  1 68 

STAINING    AND    MOUNTING    PARAFFIN    SECTIONS  -  -  170 

PART  II 
HEMATOLOGY 

ESTIMATION    OF   THE   AMOUNT   OF   H/EMOGLOBIN  -  "175 

CLINICAL   APPLICATIONS  -  -  -  -  "179 

ESTIMATION    OF   THE   RED   CORPUSCLES  -  -  -  181 

ESTIMATION    OF   THE   NUMBER    OF    LEUCOCYTES  -  "193 

THE  INVESTIGATION  OF  THE  MORPHOLOGY  OF   THE  LEUCOCYTES 

AND    RED   CORPUSCLES  -  -  -  -  -  1 98 

FIXATION  OF  BLOOD-FILMS  -----  202 
STAINING     BLOOD-FILMS     FOR    THE    INVESTIGATION    OF    THEIR 

CELLS  _.-__--  203 

DIFFERENTIAL   LEUCOCYTE   COUNT  -  -  -  -  2o6 

DIAGNOSTIC  APPLICATIONS   OF   THE   BLOOD   COUNT   AS  A  WHOLE  2l6 

PART  III 
CYTO-DIAGNOSIS 

CYTO-DIAGNOSIS  .  -  .  -  -  233 

APPENDIX  .-.----  245 

INDEX  -  -  .  -  .  247 


DESCRIPTION  OF  PLATE  I. 

Fig.  I. — Diphtheria  bacilH  (long  form)  from  a  young  culture  o 
blood-serum.     LofHer's  blue,      x   looo. 

Fig.  2. — Diphtheria  bacilli  (short  form)  and  Hoffmann's  bacillu: 
From    young    cultures    on    blood-serum.      Loffler's    blu( 

X    lOOO. 

Fig.  3. — Pneumococci  in  sputum  from  a  case  of  pneumonia 
Stained  with  dilute  carbol  fuchsin  and  thoroughly  washec 
X   1000. 

Fig.  4. — Anthrax  bacilli.  The  lower  portion  of  the  field  show 
bamboo-like  chains  and  spores,  and  is  taken  from  culture! 
Methylene  blue,  x  1000.  The  portion  showing  spores  i 
stained  with  carbol  fuchsin,  decolorized  by  brief  immersio 
in  methylated  spirit,  and  counterstained  in  methylene  blue 

Fig.  5. — Pus  showing  streptococci.  Stained  by  Gram's  methoc 
counterstained  with  eosin.      x   1000. 

Fig.  6. — Pus  showing  gonococci  and  staphylococci.  Stained  b 
Gram's  method  and  counterstained  with  dilute  carbol  fuchsir 
X  1000. 


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DESCRIPTION  OF  PLATE  II. 

Fig.  I. — Tetanus  bacilli  from  a  culture.  Stained  with  dilute 
carbol  fuchsin.      x   looo. 

Fig.  2.  —  Tubercle  bacilli  in  sputum.  Stained  with  carbol 
fuchsin  and  decolorized  in  dilute  sulphuric  acid ;  counter- 
stained  with  methylene  blue,      x   looo. 

Fig.  3. — Influenza  bacilli  in  sputum.  Some  are  contained 
within  a  leucocyte.     Loffler's  blue,      x   1000. 

Fig.  4. — Plague  bacilli  from  a  culture.  A  short  chain  and  some 
coccoid  (involution)  forms  are  shown.  Loffler's  blue. 
x   1200. 

Fig.  5. — Vibrio  of  Asiatic  cholera.  Dilute  carbol  fuchsin. 
X   1000. 

Fig.  6. — Small  colony  of  actinomyces,  as  it  appears  in  pus.  No 
clubs  are  visible.     Gram's  method,      x  600. 


PLATE  II 


I 


FicfJ 


<S 


'  s- 


ap 


J'iy^ 


*::'/ 


^^     ^ 


^     t     0  ^    •^ 


J^z^J 


Fiq  4- 


,^^ 


'HI'. 


Fiq  5 


^>y  6 


WdEE.delt. 


DESCRIPTION  OF  PLATE  III. 

Fig.  I. — Diplococciis  meningitidis  in  film,  from  cerebro-spinal  fluid 
obtained  by  lumbar  puncture  in  a  case  of  cerebro-spinal 
fever. 

Fig.  2. — Gonorrhoeal  pus,  showing  numerous  polynuclear  leuco- 
cytes, one  of  which  contains  gonococci  ;  a  part  of  a 
squamous  cell  also  shown. 

Fig.  3. — Pneumobacillus  in  pus  from  a  case  of  conjunctivitis. 

Fig.  4. — Pneumococci  in  sputum  from  a  case  of  pneumonia. 

Fig.   5. — Micrococcus  catarrhalis   in    sputum    from    a    case    of 
'    bronchitis. 

Fig.  6. — Staphylococci  in  old  and  degenerated  pus. 

This  plate  is  to  illustrate  the  main  morphological  differences 
between  the  chief  pathogenic  diplococci  and  organisms  re- 
sembling them.  All  the  films  were  stained  by  Gram  and 
counterstained  by  carbol  fuchsin.  In  Figs,  i,  2,  3,  and  5  the 
organisms  were  coloured  pink  ;  in  4  and  6  they  were  violet. 
The  figures  are  all  drawn  to  the  same  scale. 


PLATE  III. 


Si  if 


«0l"f 


Fig. 


Fig.  2. 


^  «l»?* 


Fig.  3. 


Fig.  4. 


-/  f 


^1  ^/' 


Fig.  5. 


Fig.  6. 


DESCRIPTION  OF  PLATE  IV. 

Fig.  I. — Streptothrix  (actinomycosis)  in  pus.  Many  of  the 
filaments  are  in  short  lengths,  resembling  bacilli.  From  a 
case  of  actinomycosis  of  the  pleura.     (Gram.) 

Fig.  2. — Film  from  the  tonsillar  exudate  in  Vincent's  angina 
(carbol  fuchsin),  showing  bacilli  and  spirilla. 

Fig.  3. —  Morax-Axenfeld  bacillus  in  pus  from  conjunctivitis 
(carbol  thionin). 

Fig.  4. — Microsporon  furfur  in  epidermic  scale.  Stained  by 
method  given  on  p.  96. 

Fig.  5. — Boas-Oppler  bacillus,  from  the  vomit  in  carcinoma 
ventriculi.     (Gram.) 

Fig.  6. — Bottle  bacillus  (see  p.  10 1).     (Gram.) 

(Photographs  by  Dr.  A.  Whitfield.) 


Fig.  I. 


Fig.   2. 


Fig.  3. 


Fig.  4. 


Fig.  5. 


Fig.  6. 


DESCRIPTION  OF  PLATE  V. 

Fig.   I  shows  Spirochccta  pallida  (in  the  centre)  and  Spirocha;ta 
refringens  (to  the  left),  under  very  high  magnification. 

Fig.  2  shows  the  same  organisms  under  a  yg'^^^^^  oil-immersion. 

[Lent  by  Professor  Schaudinn.) 


PLATE  V. 


\l              "    ^i^^r* '                              ' 

H^^B' 

"";    \^    * 

^^^^^B 

u 

|^^^^^^^^^^^^^^Hp# 

' 

I^^^^B'' 

^RHI' 

[ 

■f^^ 

t- 

Fig.   I. 


_^, . 

•      t  T 

• 'm , 

.    r.^ 

.' 

: 

• 

J 

Fig.  2. 


DESCRIPTION  OF  PLATE  VI. 

Fig.  I. — Megalosporon  edothrix  from  beard  region.  Hair-root 
(below)  showing  some  invasion.  Root-sheath  (above)  with 
abundant  mycehum. 

Fig.  2. — Megalosporon  endothrix  in  hair.  Notice  the  intact 
cuticle.     (Magnified  less  than  Fig.  i.) 

Fig.  3. — Megalosporon  endothrix  in  nail. 

Fig.  4. — Microsporon  Audouini  in  hair.  Spores  outside  hair 
partially  removed  to  show  mycelial  elements  within  the 
hair. 

Fig.  5. — Favus  in  hair. 

Preparations  all  stained  by  the  method  given  on  p.  96. 

{Prepared  and  photographed  by  Dr.  Whitfield.) 


PLATE  VI, 


7-    '-.>;^ 


V 


Fig.  I. 


Fig.  2. 


i 


Fig.  3. 


Fig.  4. 


Fi... 


DESCRIPTION  OF  PLATE  VII. 

Figs,  i,  2. — Lymphocytes. 

Fig.  3. — Lymphocyte  practically  devoid  of  protoplasm. 

Fig.  4. — Large  lymphocyte. 

Figs.  5,  6. — Large  hyaline  cells. 

Fig.  7. — Polynuclear  leucocytes. 

Fig.  8. — Eosinophile  leucocyte. 

Fig.  9. — Mast  cells  (these  are  from  normal  blood). 

Figs.  10,  11. — Myelocytes. 

Fig.  12. — Eosinophile  myelocyte. 

Fig.  13. — Large  mast  cell  from  a  case  of  spleno-meduUary  leuco  - 
cythaemia. 

Stained  by  Jenner  and  drawn  to  scale,  but  relatively  slightly 
larger  than  the  corpuscles  on  Plate  VIII. 


© 


Fi<jl 


PLATE    VII 


Fi^2 


Fi^  5 


Fvq  4 


Ftg  5 


FijS 


i.;rv>.>^-\ 


J<^i 


F.ff 


>\-. 


^M 


•^^«i;& 


Ftff8 


Fij,9 


:CS^rr:. 


Fi^  JO 


I'-. 

>■.:• 


.\' 


^•^l:^*^*-^' 


j-iffis 


Fiff  11 


^Vy  /J 


Wd'EE.dell. 


DESCRIPTION  OF  PLATE  VIII. 

Fig.  I. — A  normal  red  corpuscle. 

Fig.  2. — Red  corpuscle  showing  granular  basophilia. 

Fig.  3. — A  red  corpuscle  showing  polychromatophilia. 

Fig.  4. — Microcyte. 

Fig.  5.— Megalocyte. 
Fig.  6. — Poikilocytes. 

Fig.  7.— Normoblast. 

Fig.  8. — Normoblast  with  dividing  nucleus  from  a  case  of  Von 
Jaksch's  anaemia. 

Fig.  9. — Normoblast  with  vesicular   nucleus   and  polychroma- 
tophilic  stroma,  from  foetal  blood. 

Figs.   10,  ti,  12. — Megaloblasts,  the  last  showing  polychroma- 
tophil  degeneration. 

Specimens  stained  by  Jenner  and  drawn  to  scale,  appearing 
about  twice  the  size  as  when  seen  under  a  ^2"^"^^  lens  and 
IV.  eyepiece  (Leitz). 


PLATE  VIII 


Ftgl 


Fi^2 


Fi^5 


Fi<f4 


Ft^  5 


I'j 


Fi-6f6 


Fv^7 


Fvff8 


e 


F^ff9 


Fi^  lO 


Fi^  n 


Fttf  12 


Wd'EE.ddt 


DESCRIPTION  OF  PLATE  IX. 

Fig.  I. — Tuberculous  pleurisy.  Wet  preparation  stained  with 
methylene  blue,  showing  lymphocytes  and  red  corpuscles. 
(The  nucleoli  are  not  usually  so  clearly  shown,  and  are  not 
seen  in  dried  films  without  special  staining.) 

Fig.  2. — Pus  from  knee  affected  with  gonococcal  arthritis  (early 
in  the  disease).  The  figure  will  serve  to  show  the  appear- 
ances in  an  early  case  of  septic  pleurisy  or  empyema.  Poly- 
nuclear  cells  predominate,  but  there  are  a  few  lymphocytes. 
The  large  cells  are  endothelial,  and  have  ingested  some 
polynuclear  leucocytes  which  show  fragmentation  of  the 
nucleus.  Film  dried,  fixed  with  perchloride  of  mercury, 
and  stained  with  carbol  thionin. 

Fig.  3. — Pus  from  an  old  pneumococcic  empyema.  The  cells 
are  polynuclears,  but  are  highly  degenerated,  and  stain  very 
feebly.    Stained  as  in  Fig.  2.    (All  magnified  1000  diameters.) 


PLATE  IX. 


Fig.   I. 


80 


DESCRIPTION  OF  PLATE  X. 

Fig.  I. — Primary  pleurisy,  probably  due  to  true  rheumatism. 
Wet  preparation  stained  with  methylene  blue.  The  large 
cells  are  "  active "  endothelial  cells,  many  of  which  are 
crowded  with  large  refractile  granules.  Notice  the  different 
degrees  with  which  these  cells  have  taken  the  stain.  There 
are  also  some  polynuclears,  lymphocytes,  and  red  blood- 
corpuscles. 

Fig.  2. — From  a  case  of  passive  pleural  effusion  due  to  cardiac 
disease.  (The  appearances  in  that  due  to  renal  disease  are 
identical.)  Flat  ''  passive  "  endothelial  cells  often  grouped 
into  large  plates,  some  much  larger  than  those  shown.  A 
few  lymphocytes  and  red  blood-corpuscles  (the  latter  possibly 
from  the  puncture).     Film  stained  with  carbol  thionin. 

Fig.  3. — Cells  from  a  case  of  ascites  due  to  malignant  disease 
of  the  ovary.  Huge  endothelial  cells,  showing  large  clear 
vacuoles.  There  are  numerous  red  corpuscles,  one  of  which 
has  been  ingested  by  an  endothelial  cell.  Carbol  thionin 
(On  same  scale  as  Plate  IX.) 


PLATE  X. 


O 


\^ 


Fig.   I. 


CLINICAL    BACTERIOLOGY 
AND     HEMATOLOGY 

PART   I 

BACTERIOLOGY 

SECTION    I 
APPARATUS  AND  PROCESSES 

THE  BACTERIOLOGICAL  MICROSCOPE 

The  essentials  which  a   microscope   must   possess   in   order  to 
render  it  available  for  bacteriological  work  are : 

1.  K  firm  and  rigid  stand  and  stage. 

2.  A  firm,  accurate,  and  delicate  fine  adjustment. 

A  microscope  which  possesses  these  may  be  made  available  for 
bacteriological  work  by  the  addition  of  the  necessary  parts,  but 
one  that  is  deficient  in  these  respects  is  useless. 

3.  A  convex  and  a  flat  mirror. 

4.  An  Abbe's  condenser  and  iris  diaphragm. 

5.  Three  lenses,  a  low  power  [\  inch  or,  better,  |  inch),  a  high 
power  (J  inch  or  thereabouts),  and  a  y^^-inch  oil  immersion. 

If  the  practitioner  already  possesses  a  microscope  made  by  a 
reliable  firm,  and  in  good  condition,  this  should  be  sent  to  a  maker 
(not  necessarily  the  maker  of  the  microscope  in  question)  or  to  a 
bacteriologist  for  an  opinion  as  to  whether  or  no  it  is  sufficiently 
firm,  and  has  a  fine  adjustment  good  enough,  to  justify  the  addition 
of  the  other  parts.  If  this  is  the  case  it  should  be  fitted  with  an 
[Abbe's  condenser  and  an  iris  diaphragm;  the  cost  should  not 
exceed  30s.  or  £1.  But  it  is  useless  to  have  this  alteration  made 
unless  the  stand  is  sufficiently  steady  to  carry  an  oil-immersion 

I 


2  CLINICAL    BACTERIOLOGY   AND    HEMATOLOGY 

lens ;    and   in   most  cases   it   is   useless   to  think  of  having  an 
inefficient  fine  adjustment  altered  for  bacteriological  use. 

There  are  now  many  microscopes  which  are  sold  at  a  com- 
paratively small  price,  and  which  will  answer  every  purpose.  Of 
these,  Leitz's  lib.  stand,  with  the  lenses  above  mentioned,  a  triple 
nose-piece  (which  is  so  great  an  advantage  that  it  might  almost  be 
called  an  essential),  and  two  eye-pieces,  costs  about  £i^,  and  may 


Fig.  I. — Bacteriological^Microscope. 

be  highly  commended.  The  stand  is  very  firm  and  strong,  the 
fine  adjustment  delicate,  and  the  lenses  altogether  admirable.  The 
only  objection  to  this  microscope  is  that  it  is  rather  heavy  ;  this  is 
an  advantage  for  laboratory  work,  but  it  is  a  disadvantage  for  a 
medical  man,  who  may  have  to  examine  blood,  etc.,  at  the  patient's 
bedside.  Swift  and  Son  manufacture  a  microscope  of  much  the 
same  type  at  about  the  same  price  ;  it  has  all  the  good  points  of 
the  Leitz  microscope  and  is  somewhat  lighter.     The  same  firm 


THE  BACTERIOLOGICAL  MICROSCOPE  3 

also  supplies  a  portable  instrument,  the  cost  of  which  (without 
lenses)  is  £^.  This  is  very  ingeniously  made,  and  will  carry  an 
oil-immersion  lens  quite  well ;  it  is  perhaps  the  most  suitable 
stand  for  a  practitioner  who  may  have  to  do  work  at  a  distance, 
for  it  packs  into  a  very  small  compass  and  is  not  heavy.  The 
same  firm  also  make  a  microscope  designed  by  Dr.  Briscoe, 
which  is  even  more  portable,  has  a  mechanical  stage,  and  packs 
into  a  box  in  which  all  the  requisites  for  blood-counts  are  carried. 
It  is  especially  adapted  for  blood-work  at  the  bedside. 


Fig,  2. — Bacteriological  Microscope. 


The  cost  of  a  y\r-inch  oil-immersion  lens  is,  roughly  speaking, 
£^.     Beck  and  Watson  supply  good  lenses  at  £\. 

The  cost  of  the  other  lenses  may  be  put  down  at  30s.  or  less 
apiece.  Leitz's  No.  3  (about  f  inch)  costs  15s.,  and  his  No.  6 
(about  i  inch)  costs  30s. ;  these  are  both  extremely  well  adapted 
for  bacteriological  work. 

With  regard  to  eye-pieces,  it  is  an  advantage  to  have  two,  a 
No.  2  and  a  No.  4.  Higher  powers  may  be  used,  but  it  must  be 
remembered  that  any  increased  gain  in   magnification  brought 


4  CLINICAL    BACTERIOLOGY    AND    HEMATOLOGY 

about  by  the  eye-piece  is  attended  by  a  loss  of  definition.  The 
same  appUes  to  the  increased  magnification  obtained  by  pulling 
out  the  draw-tube. 


Fig.  3.— Portable  Microscope  (Swift). 


Fig.  4. — Portable  Microscope  (Swift). 

The  total  cost  of  a  microscope  may  be  put  down  at  £1^,  and 
for  this  sum  a  thoroughly  efficient  instrument  can  be  obtained, 
while  one  that  will  answer  every  purpose  may  be   bought  for 


THE  BACTERIOLOGICAL  MICROSCOPE  5 

decidedly  less.  The  cost  of  adapting  a  good  stand  will  be  about 
^6  IDS.  or  £'■]  (30S.  or  £1  for  the  substage  arrangement  and  £^  for 
the  lens),  or  less  if  a  cheaper  oil-immersion  lens  is  obtained. 

A  mechanical  stage  is  a  very  great  advantage  in  haematological 
work,  since  it  enables  a  preparation  to  be  moved  regularly  back- 
ward and  forward  and  up  and  down,  so  that  all  parts  of  its 
surface  may  be  examined  systematically.  A  simple  form  is  all 
that  is  required,  the  verniers  being  quite  unnecessary  for  most 
purposes.  It  can  be  fitted  to  most  stands.  Leitz's  can  be 
specially  recommended. 


Fig.  5. — Hot-air  Sterilizer:  Simple  Form. 

It  need  scarcely  be  said  that  there  are  many  admirable  micro- 
scopes other  than  those  mentioned  above,  which  have  simply 
been  selected  as  favourable  and  low-priced  instruments  of  English 
and  Continental  manufacture.  Amongst  others,  the  microscopes 
made  by  Beck,  Ross,  and  Watson  are  all  good,  and  reasonable 
in  price. 


STERILIZATION  OF  APPARATUS,  ETC. 

Requisites. — i.  A  thermometer  graduated  to  200°  C. 
^     2.  A  hot-air  sterilizer  ;  or  a  cubical  biscuit  tin,  the  soldering  of 
which  has  been  replaced  by  brazing — this  must  be  mounted  upon 
a  tripod  stand  ;  or  a  kitchen  oven,  preferably  a  gas  oven. 


b  CLINICAL   BACTERIOLOGY   AND    HiEMATOLOGY 

3.  A  steam  sterilizer  ;  or  a  large  kitchen  steamer — this  should 
be  deep  enough  to  contain  a  litre  flask  holding  a  funnel. 

4.  A  large  Bunsen  burner  or  spirit-lamp. 

Bacteria  and  their  spores  are  ubiquitous,  and  it  is  necessary  to 
sterilize  all  vessels  and  other  apparatus  and  all  culture  media 
before  use.  The  methods  which  are  adopted  all  depend  upon  the 
action  of  heat ;  chemical  antiseptics  are  rarely  used  in  the  bacterio- 
logical laboratory  for  the  sterilization  of  apparatus,  for  it  would  be 
difficult  to  remove  them  completely,  and  the  traces  which  might 
remain  would  prevent  the  development  of  those  germs  which  we 


Fig.  6.— Hot-air  Sterilizer. 


wished  to  cultivate.  Two  chief  methods  are  in  use,  sterilization 
by  dry  heat  and  by  steam;  we  exclude  sterilization  by  steam  under 
pressure,  as  this  requires  special  and  expensive  apparatus,  and  is 
never  absolutely  necessary,  though  often  convenient. 

Dry  Heat  is  used  to  sterilize  all  glass  vessels  (flasks,  Petri 
dishes,  test-tubes,  pipettes,  etc.),  cotton-wool,  and  metal  instru- 
ments. The  heat  must  be  continued  for  at  least  half  an  hour, 
and  must  not  fall  below  150°  C.  as  indicated  by  the  thermometer. 
Another  method,  which  is  less  reUable  than  the  use  of  the  ther- 
mometer, but  which  may  be  resorted  to  in  an  emergency,  is  to 
wrap  the  apparatus  loosely  in  cotton- wool,  and  to  proceed  with  the 


I 


STERILIZATION    OF   APPARATUS  7 

heating  (allowing  the  temperature  to  rise  gradually)  until  the  outer 
part  of  the  wool  is  slightly  singed  over  the  whole  of  the  exposed 
surface. 

The  special  sterilizer  which  is  used  in  the  bacteriological 
laboratory  consists  of  a  copper  or  iron  oven  with  double  walls 
and  perforated  metal  shelves.  There  is  a  hole  in  the  top,  which 
is  fitted  with  a  perforated  cork,  through  which  the  thermometer 
passes.  The  oven  is  mounted  on  a  stand,  and  heated  by  means 
of  a  large  Bunsen  or  Fletcher's  burner. 

An  efficient  sterilizer  may  be  made  out  of  a  cubical  biscuit  box, 
but  it  will  not  stand  much  usage  unless  the  joints  are  brazed 
instead  of  being  soldered :  this  can  be  done  by  any  tinsmith.  It 
is  much  better  to  have  the  bottom  of  the  box  replaced  by  a  sheet 
of  copper,  and  a  sterilizer  made  in  this  way  will  answer  every 
purpose  and  be  fairly  durable.  A  circular  hole  is  cut  through  the 
centre  of  the  lid  and  fitted  with  a  cork  bored  so  as  to  admit  the 
thermometer.  A  false  bottom  or  a  shelf  an  inch  or  so  from  the 
bottom  will  keep  the  articles  which  are  being  sterilized  from 
the  heated  surface  ;  the  false  bottom  may  be  made  from  a  sheet 
of  tin  2  inches  longer  in  two  of  the  sides  than  the  bottom 
of  the  box.  The  extremities  of  the  shorter  sides  are  to  be 
turned  down  for  a  length  of  an  inch,  and  several  holes  cut  in 
the  plate. 

Lastly,  the  kitchen  oven  may  be  pressed  into  service  if  no 
other  sterihzer  is  at  hand  in  an  emergency.  The  apparatus  to  be 
sterilized  is  to  be  placed  on  a  layer  of  cotton-wool  on  one  of  the 
shelves,  and  the  temperature  is  observed  by  means  of  the  thermo- 
meter, which  should  be  thrust  through  the  little  window  which 
permits  of  the  regulation  of  the  temperature  ;  or  the  heat  may 
be  continued  until  the  cotton-wool  is  singed  over  the  whole  of 
the  exposed  surface.  This  method  is  very  convenient  for  prac- 
titioners sending  materials  to  a  laboratory  for  bacteriological 
examination. 

A  gas  oven  is  even  more  convenient,  as  the  temperature  can 
be  regulated  to  a  nicety. 

All  glass  apparatus  must  be  thoroughly  cleansed  and  dried 
before  sterilization.  The  remaining  steps  differ  somewhat  in  the 
different  cases. 

Flasks  are  plugged  lightly  with  cotton-wool  before  being  placed 
in  the  sterilizer.  Bottles  may  be  sterilized  in  the  same  way ; 
they  may  also  be  sterilized  by  boiling.     Test-tubes  are  treated  in 


8 


CLINICAL    BACTERIOLOGY   AND    HEMATOLOGY 


the   same  way  as  flasks.     Petri  dishes  are  wrapped  round  with 
tissue-paper  or  filter-paper  before  being  sterihzed. 

Before  removing  glass  apparatus  from  the  sterilizer  remember 
to  let  the  temperature  fall  gradually,  or  the  vessels  may  crack. 

If  a  sterilized  test-tube  be  required  in  a  hurry  (as  often 
happens),  plug  a  clean  tube  with  cotton-wool,  and  hold  it  with 
forceps,  one  blade  being  inside  and  one  outside  the  open  end. 
Then  heat  every  part  of  the  tube  thoroughly  in  the  flame,  taking 
care  the  heat  is  great  enough  to  scorch  the  plug. 

Cotton-wool  is  sterilized  by  being  spread  out  in  thin  layers  on 

the  shelves  of  the  apparatus,  and 
the  heat  is  continued  until  the  out- 
side is  singed. 

Metal  instruments  (knives,  scissors, 
etc.)  may  be  sterilized  in  the  same 
way  and  at  the  same  time.  They 
should  be  wrapped  loosely  in  cotton- 
wool, and  should  not  be  removed 
from  their  wrapping  until  the  mo- 
ment at  which  they  are  to  be  used. 

Steam  is  chiefly  used  for  the 
sterilization  of  culture  media  before 
use,  and  for  the  destruction  of  cul- 
tures when  they  are  done  with.  The 
latter  purpose,  however,  is  accom- 
plished more  speedily  and  safely  by 
the  addition  of  a  few  drops  of  com- 
mercial formalin  to  each  tube,  or 
the  tubes  may  be  filled  with  i  in  20 
carbolic,  which,  however,  cannot  be 
relied  on  to  kill  spores. 
The  proper  steam  sterilizer  consists  of  a  metal  cylinder  with  a 
perforated  diaphragm  6  or  8  inches  from  the  bottom.  It  is  en- 
closed in  a  thick  layer  of  felt  or  other  non-conductor  of  heat,  and 
is  provided  with  a  lid.  The  space  between  the  bottom  and  the 
diaphragm  is  partly  filled  with  water,  which  is  boiled  by  means  of 
a  Bunsen  flame  or  Fletcher's  burner,  the  apparatus  to  be  sterilized 
being  placed  in  the  chamber  above  so  as  to  be  exposed  to  the 
steam. 

An  ordinary  steamer  (such  as  is  used  for  cooking  potatoes  or 
fish)  will  answer  every  purpose.     In  procuring  such  a  steamer 


Fig.  7. — Steam  Sterilizer. 


PREPARATION  OF  CULTURE  MEDIA  9 

for  bacteriological  use  it  is  best  to  choose  one  that  will 
accommodate  a  litre  flask  holding  a  funnel,  as  it  is  often  a 
great  convenience  in  the  filtration  of  fluids  which  become 
solid  on  cooling  to  carry  out  the  process  in  an  atmosphere  of 
steam. 

Exact  details  of  the  way  it  is  used  will  be  given  subsequently. 

PREPARATION  OF  CULTURE  MEDIA 

Bacteria  are  grown  in  the  same  way  as  other  plants.  A 
gardener  who  wishes  to  grow  a  plot  of  a  particular  plant  will  first 
prepare  a  soil  suitable  for  the  growth  of  that  plant,  and  free  it  as 
far  as  possible  of  all  seeds,  roots,  etc.  He  will  then  sow  it  with 
the  seeds  of  the  plant  in  question,  and  do  what  he  can  to  expose 
them  to  a  suitable  temperature.  An  exactly  similar  process  is 
adopted  when  we  wish  to  cultivate  the  smallest  of  all  plants.  The 
soil  which  we  prepare  is  called  the  culture  medium,  and  differs  in 
the  case  of  different  bacteria ;  the  process  of  freeing  this  soil  from 
bacteria  and  their  spores  is  called  sterilization,  and  we  insure  a 
suitable  temperature  by  means  of  an  incubator,  the  heat  of  which 
is  kept  constant. 

The  culture  media  which  are  used  for  special  purposes  are 
almost  innumerable,  but  in  the  daily  routine  of  the  laboratory 
and  for  diagnostic  purposes,  broth,  gelatin,  agar-agar,  and 
blood-serum,  are  all  that  are  really  necessary  in  the  vast 
majority  of  cases.  These  media  may  all  be  bought  from  any 
firm  of  manufacturing  chemists,  or  from  any  bacteriological 
laboratory ;  and  their  purchase  saves  a  great  deal  of  work, 
and  is  to  be  recommended  for  those  who  only  wish  to  use 
them  occasionally.  They  are  sold  in  test-tubes,  which  are  kept 
sterile  by  being  plugged  fairly  firmly  with  cotton-wool ;  this  sub- 
stance prevents  the  passage  of  bacteria  as  long  as  it  is. kept  dry. 
The  tubes  are  best  stored  in  jars  provided  with  tightly  fitting  lids, 
and  it  is  an  advantage  to  place  a  shallow  layer  of  a  solution  of 
perchloride  of  mercury  (or  other  non-volatile  antiseptic)  in  the 
bottom  of  each  jar  to  prevent  the  medium  from  drying  up.  It  is 
scarcely  necessary  to  add  that  not  the  smallest  trace  of  the  lotion 
should  be  allowed  to  come  into  contact  with  the  cotton-wool 
plug  of  the  tubes.  Or  the  medium  may  be  kept  from  drying 
up  by  covering  the  tubes  with  indiarubber  caps  sold  for  the 
purpose. 


10  CLINICAL    BACTERIOLOGY   AND    HEMATOLOGY 

Broth  is  very  easily  made,  and,  as  it  is  the  foundation  of  many 
other  media,  the  practitioner  is  strongly  advised  to  prepare  it  for 
himself. 

Requisites. — i.  Liebig's  Extract  of  Meat. 

2.  Peptone. 

3.  Common  salt. 

4.  A  dilute  solution  of  sodium  carbonate — about  i  per  cent., 
but  the  exact  strength  does  not  matter. 

5.  A  large  flask,  a  stirring-rod,  and  a  large  glass  funnel. 

6.  Test-tubes  and  cotton-wool  plugs.  The  exact  size  of  the 
tubes  is  unimportant,  but  6  inches  by  f  inch  is  convenient.  The 
plugs  are  best  prepared  from  wool  which  has  been  previously 
sterilized  by  dry  heat,  and  should  be  fairly  firm.  The  tube,  with 
the  plug  in  situ,  must  be  sterilized  by  dry  heat  ready  for  use. 

7.  Litmus-paper. 

Method. — Take  i  litre  of  tap-water  in  the  flask,  and  add 
5  grammes  of  Liebig's  Extract,  10  grammes  of  peptone,  and 
5  grammes  of  common  salt,  and  boil  until  all  are  dissolved.  Test 
the  reaction  by  withdrawing  a  drop  of  the  fluid  on  the  stirring-rod 
and  applying  it  to  a  piece  of  litmus-paper.  You  will  probably 
find  that  it  is  slightly  acid.  Now  add  some  of  the  solution  of 
soda,  drop  by  drop,  testing  after  each  addition,  until  the  reaction 
of  the  fluid  is  slightly  alkaline.*  Boil  the  fluid  for  half  an  hour, 
to  coagulate  any  albumin  which  may  be  present. 

Next  filter  the  broth  into  a  sterile  flask,  passing  it  through  a 
double  thickness  of  white  filter  or  blotting  paper,  and  plug  the 
flask  firmly  with  sterilized  cotton-wool. 

If  the  broth  is  to  be  used  for  the  manufacture  of  gelatin  or 
agar,  it  is  next  sterilized  in  the  flask ;  while  if  it  is  to  be  used 
as  it  is  as  a  culture  medium,  it  is  decanted  into  tubes  and  then 
sterilized. 

In  decanting  media  into  tubes  be  very  careful  not  to  .get  the 
plug  wet,  and  not  to  let  any  of  the  medium  get  on  to  the  upper 
part  of  the  tube  ;  otherwise  the  plug  will  stick  to  the  tube,  and 
there  will  be  some  danger  of  bacteria  from  the  air  "  growing 
through "  the  fluid  contained  in  the  interstices  of  the  plug  and 
contaminating  the  culture.    Ordinary  non-absorbent  (brown)  wool 

*  If  during  the  neutralizing  process  too  much  alkali  is  added,  then  it  is 
necessary  to  reacidify  with  dilute  hydrochloric  acid  and  reneutralize.  The 
sodium  chloride  formed  makes  no  practical  difference  in  the  medium. 


i 


PREPARATION    OF   CULTURE    MEDIA  II 

is   better  than   the   white  absorbent   wool,    as   it   is   less   easily 
wetted. 

The  broth  (and  other  culture  medium  after  being  melted)  may 
be  poured  into  the  tubes  in  the  following  way  :  A  sterilized  funnel 
is  united  by  a  short  length  of  indiarubber  tubing  to  a  piece  of  glass 
tubing  drawn  out  to  a  point ;  the  rubber  tube  is  clipped  by  a 
spring  clip  or  a  pair  of  pressure  forceps.  The  funnel  is  now 
mounted  on  a  retort-stand,  filled  with  the  medium,  and  covered 
over  with  a  piece  of  glass.  The  cotton-wool  plug  is  removed  from 
a  test-tube,  and  the  latter  placed  so  that  the  glass  tube  attached  to 
the  funnel  reaches  nearly  to  the  bottom.  The  clip  is  released,  and 
the  requisite  quantity  of  broth  (enough  to  fill  the  tube  to  the  depth 
of  if  or  2  inches)  is  allowed  to  run  in  ;  the  clip  is  then  reapplied 
and  the  tube  removed  and  plugged.  This  process  is  repeated 
until  enough  tubes  have  been  filled. 

The  tubes  and  the  broth  which  remains  over  (after  having  been 
poured  back  into  the  flask  and  the  latter  plugged  with  cotton-wool) 
are  now  sterilized.  The  vessels  are  placed  in  the  steam  sterilizer 
and  exposed  to  steam  for  half  an  hour  on  three  successive  days  ;  this 
process  is  called  intermittent  sterilization,  and  its  rationale  is  very 
simple.  The  first  steaming  destroys  all  developed  bacteria,  and 
would  sterilize  the  fluid  entirely  if  no  spores  were  present.  In 
the  interval  between  the  first  and  second  sterilization  most  of  the 
spores  which  may  be  present  will  develop  into  mature  bacteria, 
and  these  will  be  killed  by  the  second  steaming.  The  third 
sterilization  is  to  kill  off  any  bacteria  which  may  not  have 
developed  from  spores  in  the  first  interval.  A  very  similar 
process  is  adopted  by  the  gardener  in  freeing  soil  from  weeds  ; 
the  application  of  chemical  weed-destroyers  or  a  thorough  hoeing 
will  destroy  developed  plants,  but  will  not  injure  seeds  which  may 
be  contained  in  the  soil,  and  these  processes  are  repeated,  intervals 
being  allowed  to  permit  the  development  of  the  plants,  until  they 
reach  the  stage  in  which  they  are  vulnerable. 

To  recapitulate  :  Mix  the  ingredients,  and  heat  until  they  are 
dissolved,  render  slightly  alkaline,  boil  half  an  hour,  filter.  Then 
place  in  sterilized  flask  or  into  test-tubes,  and  sterilize  in  the  steam 
sterilizer  for  half  an  hour  on  three  successive  days. 

Nutrient  Gelatin  is  broth  which  has  been  solidified  by  the 
addition  of  from  lo  to  15  per  cent,  of  gelatin  ;  the  former  amount 
is  used  in  the  winter,  the  latter  in  the  summer.  For  general 
purposes  12^  per  cent,  may  be  used  in  all  cases. 


12  CLINICAL    BACTERIOLOGY    AND    HEMATOLOGY 

The  Special  advantages  of  gelatin  as  a  culture  medium  are  two- 
fold. In  the  first  place,  a  gr^t  many  organisms  grow  in  or  on  it 
in  a  characteristic  way,  so  that  a  bacteriologist  may  be  able  to 
identify  the  organism  by  inspection  of  the  culture.  This  arises 
partly  from  the  fact  that  some  bacteria  produce  a  ferment  which 
digests  gelatin  just  as  pepsin  does  ;  these  bacteria  "  liquefy  "  the 
gelatin,  and  the  distinction  between  the  bacteria  which  have  and 
those  which  have  not  this  property  is  very  important  for  purposes 
of  diagnosis.  Further,  some  bacteria  liquefy  rapidly  and  others 
slowly,  and  this  is  another  important  point  in  the  identification  of 
a  germ. 

In  the  second  place,  the  gelatin  medium  may  be  melted  at  a 
temperature  (about  25°  C.)  at  which  bacteria  are  not  killed.  This 
fact  is  made  use  of  in  the  isolation  of  bacteria  from  a  fluid  which 
contains  several  species  by  the  process  known  as  "  plating." 
Suppose,  for  instance,  that  we  find  by  microscopic  examination 
that  a  specimen  of  pus  contains  two  different  species  of  bacteria 
(perhaps  a  bacillus  and  a  coccus),  and  we  wish  to  obtain  the  two 
organisms  in  pure  culture  so  that  we  can  ascertain  their  nature 
and  properties.  We  take  a  tube  of  gelatin  and  melt  it  by  placing 
it  in  warm  water,  and  then  inoculate  the  medium  with  a  minute 
quantity  of  the  pus.  We  then  shake  it  so  as  to  distribute  the 
organisms  throughout  the  melted  fluid,  and  then  pour  the  latter 
into  a  flat  dish  (Petri's  plate),  so  that  the  gelatin  flows  out  into  a 
thin  film  and  then  sets.  If  our  dilution  has  been  properly  made, 
we  shall  have  separated  each  organism  from  its  neighbours,  and 
each  separate  germ  will  grow  up  into  a  "colony,"  which  will  soon 
be  visible  to  the  naked  eye.  In  all  probability  we  shall  be  able 
to  see  that  these  colonies  are  of  two  kinds :  one  may  liquefy 
and  the  other  not,  one  may  be  coloured  and  the  other  colour- 
less, one  may  be  round  and  the  other  angular,  etc.  Samples 
of  each  sort  of  colony  are  then  transplanted  to  fresh  culture- 
tubes,  and  again  incubated.  An  example  of  this  process  is  given 
on  p.  58. 

A  slight  modification  of  this  process  enables  us  to  make  an 
estimate  of  the  number  of  living  bacteria  which  is  present  in  a 
given  fluid.  To  do  this  we  have  to  follow  out  the  above  process, 
adding  a  definite  measured  quantity  of  the  fluid  to  the  culture-tube 
of  liquefied  gelatin.  The  number  of  colonies  which  develop  is 
counted,  and  this  gives  us  the  number  of  bacteria  in  the  sample  of 
fluid.      For   example,  if    ^^   c.c.  diff"used   throughout  a  tube  of 


I 


PREPARATION    OF    CULTURE    MEDIA  I3 

melted  gelatin  and  poured  out  into  a  thin  film  produced  twenty 
colonies,  it  follows  that  i  c.c.  of  the  fluid  contained  200  bacteria. 
This  is  a  brief  description  of  the  essentials  of  the  method  adopted 
in  the  quantitative  examination  of  water  and  other  fluids. 
Requisites  for  the  Mamifacttive  of  Gelatin. — i.  Broth. 

2.  Gelatin.  (Coignet's  gold  label  gelatin  is  best,  but  any  good 
brand  will  do.) 

3.  Dilute  solution  of  sodium  carbonate. 

4.  Litmus-papers. 

5.  Flasks,  stirring-rod,  funnel,  and  plugged  test-tubes  as  for 
broth. 

Method. — Measure  the  broth  and  add  to  it  12 J  grammes  of 
gelatin  for  each  100  c.c.  ;  allow  to  soak  for  an  hour  or  more,  and 
then  heat  until  the  gelatin  is  dissolved.  Continue  the  heat,  and 
render  the  medium  faintly  alkaline,  just  as  was  done  in  the 
preparation  of  broth.  Now  filter  through  a  moistened  filter-paper. 
To  avoid  the  setting  of  the  gelatin  during  the  filtration,  it  is  best 
to  use  a  double-jacketed  funnel  containing  hot  water,  but  if  this  is 
not  at  hand  the  whole  apparatus  (flask  and  funnel)  may  be  placed 
in  the  steam  sterilizer  (the  lid  being  kept  off  to  avoid  the  drops 
of  condensed  water  which  might  otherwise  fall  into  the  funnel) 
or  in  a  wavm  (but  not  hot)  oven,  and  left  at  a  temperature  of  about 
40°  C,  until  the  process  is  complete. 

The  gelatin  which  is  made  by  the  above  process  is  sufficiently 
clear  for  most  purposes.  A  more  sightly  medium  may  be  made 
by  clarification  of  the  above  by  white  of  Qgg.  To  the  medium 
(after  neutralization,  but  before  filtration)  add  the  white  of  one 
egg  for  each  250  or  300  c.c.  of  fluid,  and  shake  thoroughly.  Now 
boil  in  the  steamer  for  half  an  hour,  and  filter  as  before. 

Test-tubes  are  filled  with  gelatin  just  in  the  same  way  as  with 
broth,  and  the  process  must  be  carried  out  quickly  to  avoid 
solidification  of  the  medium.  Some  of  the  test-tubes  are  allowed 
to  cool  in  the  vertical  position,  others  lying  in  a  sloping  position, 
so  that  the  upper  surface  of  the  gelatin  forms  an  ellipse  some 
3  inches  long.  The  former  tubes  are  inoculated  by  driving  a 
straight  platinum  needle  charged  with  the  material  containing 
the  bacteria  into  the  gelatin  in  the  axis  of  the  tube ;  cultures 
made  in  this  way  are  called  "stab  cultures."  The  gelatin 
"  slopes  "  are  inoculated  by  drawing  the  charged  needle  along  the 
surface  of  the  medium,  care  being  taken  not  to  plough  it  up ; 
cultures  made  in  this  way  are  called  "  stroke  cultures." 


14  CLINICAL    BACTERIOLOGY    AND    HEMATOLOGY 

Agar,  or  Agar-Agar,  is  the  name  given  to  the  dried  strips  of  a 
Japanese  seaweed.  It  forms  a  jelly  which  differs  from  that  con- 
taining gelatin  in  that  it  melts  at  a  higher  temperature ;  nutrient 
agar,  as  used  in  the  laboratory,  melts  just  below  the  boiling-point 
of  water  and  sets  at  about  40°  C.  This  is  an  advantage  in  the 
cultivation  of  most  pathogenic  bacteria,  for  these  grow  (as  a  rule) 
best  at  or  near  the  temperature  of  the  body,  the  temperature  to 
which  they  are  exposed  under  natural  circumstances  ;  and  at  this 
temperature  gelatin  would  melt.  Agar  is  somewhat  difficult  to 
prepare  unless  the  practitioner  has  an  autoclave,  and  may  be 
bought  with  advantage.  But  the  following  method  is  not  very 
difficult,  and,  as  agar  is  perhaps  the  most  generally  useful  of  all 
media,  should  be  learnt. 

Requisites. — i.   Broth. 

2.  Agar-agar.  This  should  be  cut  up  into  very  small  pieces  with 
a  pair  of  scissors,  or  may  be  bought  in  powder. 

3.  Solution  of  acetic  acid  (glacial  acetic  acid,  2  to  4  c.c. ;  water, 
500  c.c). 

4.  A  large  beaker. 

5.  Other  apparatus  and  materials  as  for  gelatin. 

Method. — Weigh  out  2  grammes  of  agar  for  each  100  c.c.  of 
broth  to  be  used,  and  soak  it  in  the  dilute  acetic  acid  for  a  quarter 
of  an  hour.  Now  strain  off  the  acid  and  wash  the  agar  in  water 
until  a  small  piece  does  not  redden  blue  litmus-paper  when  pressed 
upon  it.  Place  the  broth  in  a  glass  beaker  and  add  the  agar. 
Now  place  the  beaker  upon  a  piece  of  wire  gauze  on  a  tripod 
stand,  and  apply  a  small  Bunsen  flame  or  spirit-lamp ;  this  must 
be  placed  so  that  the  flame  impinges  on  a  point  not  far  from  the 
side  of  the  beaker.  As  the  fluid  is  heated  it  will  rise,  and  a 
continual  circulation  will  take  place,  so  that  the  fragments  will 
not  stick  to  the  bottom  and  cause  it  to  crack.  When  all  is 
dissolved,  the  hot  liquid  must  be  carefully  neutralized.  It  is  then 
allowed  to  cool  to  about  50°  C,  and  the  white  of  an  egg  added  for 
each  500  c.c.  of  fluid  and  mixed  in  thoroughly  by  being  stirred 
with  a  glass  rod.  The  whole  is  then  placed  in  the  steamer  for  an 
hour,  at  the  end  of  which  time  the  albumin  should  be  completely 
coagulated.  The  beaker  and  its  contents  are  then  allowed  to  cool 
gradually,  so  that  the  coagulum  (retaining  all  solid  particles)  may 
settle  to  the  bottom  before  coagulation  is  complete.  Perhaps  the 
best  method  of  accomplishing  this  is  to  place  it  in  the  oven 
(taking  care  that  the  temperature  does  not  exceed  100°  C.)  after 


PREPARATION    OF   CULTURE    MEDIA  15 

the  lire  has  been  raked  out  at  night.  In  the  morning  the  mass 
will  be  found  to  have  soUdified,  and  there  will  be  a  coagulum  at 
the  bottom.  The  beaker  is  then  inverted  and  the  mass  "  turned 
out "  just  as  a  cook  turns  out  a  jelly,  and  the  sediment  is  cut 
off  with  a  sharp  knife.  This  avoids  filtration,  which  is  very 
troublesome. 

An  alternative  method  is  to  filter  the  melted  jelly  through 
moistened  filter-paper.  It  is  necessary  to  keep  flask  and  funnel  in 
a  steamer  (the  water  of  which  is  kept  boiling  vigorously)  during 
the  whole  process,  or  the  jelly  will  solidify  in  the  outflow  tube  of 
the  funnel.  Or  it  may  be  filtered  through  a  double  thickness  of 
ordinary  surgical  lint  (non-medicated).  It  runs  through  this  very 
quickly,  and  the  funnel  need  not  be  kept  hot.  The  resulting 
medium  is  not  absolutely  clear,  but  sufficiently  so  for  most 
purposes. 

The  agar  is  again  melted  and  placed  in  test-tubes ;  these  are 
sterilized  on  three  successive  days  and  allowed  to  set  in  a  sloping 
position.  For  certain  purposes  glucose,  glycerin,  etc.,  are  added 
to  the  agar.  The  addition  should  be  made  to  the  melted  medium 
just  before  it  is  poured  into  the  tubes. 

Solidified  Blood-serum  is  very  difficult  to  prepare,  and  is 
best  purchased  ready  for  use  from  a  good  laboratory.  It  is  used 
chiefly  in  the  diagnosis  of  diphtheria  by  the  examination  of 
''  swabs  "  from  the  throat. 

Potato  tubes  are  in  occasional  use,  and  are  easy  to  prepare. 
The  process  is  as  follows  :  Take  large  and  sound  potatoes  and 
scrub  them  thoroughly  with  a  nail-brush  under  the  tap.  Peel 
them  deeply  enough  to  remove  the  eyes  completely.  Then  cut 
them  into  cylinders  a  little  less  than  j  inch  in  diameter  (if  you  are 
using  |-inch  test-tubes)  and  as  long  as  possible ;  this  is  best  done 
by  means  of  a  cork-borer,  but  they  may  be  shaped  by  means  of  a 
knife  if  this  is  not  at  hand.  Then  cut  each  cylinder  in  half  by  a 
cut  running  obliquely  from  end  to  end  ;  the  shape  of  each  half 
should  be  exactly  like  that  of  the  medium  in  a  sloped  gelatin  tube. 
Place  the  halves  in  a  large  vessel  of  tap-water  and  allow  them 
to  soak  all  night ;  it  is  a  good  plan  to  use  running  water  if 
possible. 

After  this  has  been  done  place  each  half  (base  downwards)  in  a 
test-tube,  having  previously  inserted  a  small  mass  of  absorbent 
cotton-wool  and  enough  water  to  saturate  it.  Plug  the  mouth  of 
the  tube  with  cotton-wool  and  sterilize  on  three  successive  days. 


l6  CLINICAL    BACTERIOLOGY   AND    HiEMATOLOGY 


INOCULATION   OF   CULTURE   MEDIA 

The  method  in  which  this  is  done  varies  greatly  according  to 
the  end  in  view,  and  variations  of  the  process  now  to  be  described 
will  be  mentioned  under  their  appropriate  headings.  We  will 
suppose  that  we  have  to  examine  a  specimen  of  pus,  and  wish  to 
make  a  stroke  culture  on  agar  and  a  stab  culture  in  gelatin.  The 
following  must  be  at  hand  : 

1.  The  pus. 

2.  A  sloped  agar  tube  and  a  stab  gelatin  tube. 

3.  A  Bunsen  burner  or  a  spirit-lamp  with  a  tall  flame. 

4.  A  pair  of  dissecting  forceps. 

5.  Platinum  needles.  Each  needle  consists  of  a  piece  of 
platinum  wire  about  3  inches  long  mounted  in  the  axis  of  a  glass 


Fig.  8. — Platinum  Needles. 

rod  about  6  or  8  inches  in  length.  The  wire  should  be  just  thick 
enough  not  to  bend  too  easily.  They  are  easily  prepared.  The 
rod  is  selected,  and  the  length  of  platinum  wire  is  held  in  an 
ordinary  pair  of  forceps.  The  end  of  the  glass  rod  is  held  in  the 
flame  until  quite  soft ;  the  end  of  the  wire  is  then  heated  to 
redness,  and  pushed  into  the  rod  to  the  depth  of  about  J  inch, 
taking  care  that  it  is  kept  in  the  axis.  The  whole  is  allowed 
to  cool,  and  is  ready  for  use. 

For  some  purposes  we  use  needles  which  terminate  in  a  small 
loop,  so  that  they  will  retain  a  drop  of  fluid.  These  are  prepared 
in  the  same  way  as  the  straight  needles,  the  free  end  of  the  wire 
being  subsequently  twisted  round  a  French  nail  or  other  suitable 
object. 

The  method  is  as  follows : 

I.  Hold  the  culture-tube  you  are  going  to  inoculate  first  between 
the  index  and  middle  fingers  of  the  left  hand,  pointing  the  mouth 


INOCULATION    OF   CULTURE    MEDIA  17 

of  the  tube  slightly  downwards  (so  that  no  dust  shall  drop  into 
it)  and  to  the  right.  Tubes  of  solid  media  should  always  be  held 
in  this  position  during  inoculation  ;  tubes  of  liquid  media  are  held 
in  a  similar  way,  but  of  course  their  mouths  must  point  upwards. 

2.  Put  the  projecting  portion  of  the  cotton-wool  plug  of  the  test- 
tube  into  the  flame  so  as  to  singe  it ;  this  is  to  destroy  any  germs 
which  may  have  been  deposited  upon  it  from  the  atmosphere. 

3.  SteriHze  the  points  of  the  forceps  by  passing  them  slowly 
through  the  flame,  and  then  use  them  to  remove  the  plug.  Place 
this  between  the  ring  and  little  fingers  of  the  left  hand,  and  put 
the  forceps  down. 

4.  Take  the  platinum  needle  in  the  right  hand,  heat  the  whole 
of   the  wire  to   redness,  and   pass   the   lower  3  or  4   inches   of 


Fig.  9. 

the  glass  rod  slowly  through  the  flame.  Remember  that  every 
portion  of  the  needle  which  goes  inside  the  tube  must  be  sterilized 
in  this  way.  Allow  the  needle  to  cool ;  you  should  have  found 
out  how  long  this  will  take  by  a  previous  experiment. 

5.  Dip  the  tip  of  the  needle  into  the  pus  ;  pass  it  into  the  tube 
until  it  reaches  nearly  to  the  bottom  of  the  tube  (now  uppermost), 
and  allow  it  to  rest  upon  the  sloping  surface  of  the  medium ;  now 
withdraw  it  gently,  allowing  the  tip  of  the  wire  to  trail  gently 
along  the  whole  length  of  the  sloped  surface.  Do  not  touch  the 
medium  with  the  glass  shoulder  of  the  needle. 

6.  Sterilize  the  needle  as  before.  This  step  must  never  be 
forgotten. 

7.  Take  the  cotton-wool  plug  in  the  forceps,  put  it  in  the  flame, 
and  singe  all  parts  of  its  surface.  Then  plug  the  tube  while 
the  wool  is  still  burning.     Label  it. 

To  make  a  stab  culture  take  the  other  gelatin  tube  and  proceed 
as  before  until  you  get  to  step  5.     When  you  have  passed  the 

2 


l8  CLINICAL    BACTERIOLOGY   AND    HEMATOLOGY 

needle  into  the  tube  drive  it  steadily  into  the  medium,  taking  care 
not  to  deviate  from  the  axis  of  the  tube.  Finish  the  process  as 
before. 

All  this  may  seem  involved.  As  a  matter  of  fact  it  is  very 
simple,  and  need  not  take  more  than  a  minute  to  perform.  But 
every  step  must  be  carried  out,  and  the  whole  process  must  be 
learnt  so  thoroughly  that  it  is  performed  automatically  whenever 
a  culture  is  made. 


INCUBATION    OF    CULTURES 

The  limits  between  which  bacteria  can  live  are  very  wide  ; 
some  grow  best  at  one  temperature,  others  at  another,  the  limits 
for  the  great  majority  of  organisms  being  about  i6°  C.  and  40°  C. 
In  practice  two  temperatures  are  all  that  is  used  for  ordinary 
work.  The  lower,  or  so-called  "  room  temperature,"  is  about 
20°  C.  (68°  F.),  and  is  of  most  use  for  those  bacteria  which  grow 
naturally  outside  the  body — i.e.,  as  saprophytes.  The  higher,  or 
body  temperature,  is  about  37°  C.  (98-6  F.),  and  is  the  best 
temperature  for  the  majority  of  germs  which  live  within  the 
body — i.e.,  the  parasites.  It  is  obvious  that  gelatin  cannot  be 
incubated  at  this  high  temperature,  as  it  melts  at  25°  C.  or 
thereabouts  ;  but  all  other  media  are  available. 

The  term  "  room  temperature  "  must  not  mislead  the  practi- 
tioner, for  the  temperature  of  many  rooms  is  not  constant  at  or 
near  20°  C.  for  periods  sufficiently  long  to  permit  of  its  use  for 
incubating  bacteria.  In  the  laboratory  we  use  an  incubator,  the 
temperature  of  which  is  regulated  by  means  of  an  automatic 
regulator,  and  remains  constant  for  long  periods  whatever  be  the 
external  temperature.  It  is  hardly  necessary  for  the  practitioner 
to  purchase  one  of  these.  Careful  search  in  the  house  will 
usually  reveal  some  cupboard  or  corner  in  which  the  temperature 
will  remain  sufficiently  near  20°  C.  for  a  sufficiently  long  period ; 
it  is  more  important  that  it  should  not  rise  above  22°  C.  than  that 
it  should  not  fall  below  18°  C,  as  the  former  temperature  may 
melt  the  gelatin,  while  the  latter  will  only  delay  the  growth  of  the 
colonies.  It  will  probably  be  necessary  to  find  one  such  place  in 
the  hot  weather  {e.g.,  the  cellar)  and  another  one  in  the  winter 
{e.g.,  a  cupboard  not  far  from  the  hot-water  pipes). 

It  is  necessary  that  cultures  which  are  being  incubated  should 


INCUBATION    OF   CULTURES 


19 


be  kept  in  the  dark,  as  light  is  inimical  to  the  development  of 
nearly  all  bacteria. 

Incubation  at  the  body  temperature  presents  more  difficulty. 
An  incubator  is  almost  essential  when  much  work  has  to  be  done, 
and  of  these  Hearson's  is  by  far  the  best.  The  smallest  size  costs 
about  £6  los.,  and  is  a  thoroughly  reliable  and  durable  affair. 
Cheaper  instruments  are  also  procurable:  the  "Edinburgh" 
incubator  (Alex.  Fraser,  22,  Teviot  Place,  Edinburgh)  costs  about 


Fig.  10. — Incubator. 

£^  I  OS.  complete.    Foreign  incubators  can  be  obtained  at  an  even 
lower  price,  but  are  not  very  durable. 

Much  can  be  done  without  the  use  of  so  expensive  an  apparatus 
if  the  practitioner  can  find  a  room  in  which  the  temperature  keeps 
approximately  constant  throughout  the  twenty-four  hours.  A  tin 
biscuit  box  (or  any  other  metal  box)  is  covered  with  cotton-wool 
on  the  top  and  sides,  the  bottom  being  left  bare,  and  mounted  on 
a  tripod  stand.     It  is  heated  by  means  of  an  ordinary  night-light 

2 — 2 


20 


CLINICAL    BACTERIOLOGY   AND    H.EMATOLOGY 


(two  may  be  necessary  if  the   weather   is   cold)  shielded  from 

draughts  by  means  of   a  wide  lamp-chimney  or  a   tin  cylinder 

made  out  of  an  ordinary  canister.     The  temperature  is  observed 

by  means  of  a  thermometer  projecting  through  a  hole  in  the  lid, 

and  the  night-light  raised  or  lowered  until  the  temperature  reaches 

the  desired  figure.     The  whole  apparatus  should  be  placed  on  a 

metal   tray   containing   a   small   quantity  of 

Q  water,  and  put  in  the  middle  of  the  floor,  and 

B  away  from  any  inflammable  materials.     This 

yiz^l — Ti       will  be  found  to  answer  admirably,  and  can 

y^     /^         easily  be  fitted  up  in  an  emergency. 

It  would  be  better  to  use  a  tin  box  specially 
made  for  the  purpose,  and  having  a  door  at 
the  side  and  a  perforated  false  bottom,  so  that 
the  culture-tubes  do  not  rest  directly  on  the 
metal  exposed  to  the  flame.  This  latter  had 
best  be  made  of  copper. 

Dr.  Bottomley,  of  Boscombe,  informs  me 
that  he  has  used  the  simple  apparatus  shown 
in  Fig.  1 1  for  five  years,  and  finds  it  answers 
admirably  in  the  diagnosis  of  diphtheria,  etc. 
It  is  an  ordinary  square  vaseline  tin,  into  the 
round  opening  of  which  is  put  a  cylindrical  coffee  tin.  The 
outer  tin  is  filled  with  water,  and  a  thermometer  is  put  into  a  hole 
through  its  lid.  The  culture-tubes  are  placed  in  the  inner  tin,  and 
the  apparatus  is  heated  by  a  paraffin  lamp. 

If  the  practitioner  is  fortunate  enough  to  possess  a  conservatory 
which  is  kept  at  a  temperature  approximating  to  that  of  the  body, 
this  will  serve  admirably.  The  culture-tubes  must  be  kept  in  a 
box  which  will  exclude  light. 

In  the  absence  of  this  a  cupboard  near  the  kitchen  fire  or  the 
hot-water  cistern  may  be  found  that  will  answer  the  purpose ;  a 
thermometer  should  be  placed  in  it,  and  examined  from  time  to 
time  throughout  the  day,  and  if  the  temperature  does  not  fall 
below  30°  nor  rise  above  40°,  it  will  serve  at  a  pinch,  though  a 
temperature  which  is  more  constant  near  37°  is  desirable.  It  has 
to  be  remembered  that  we  are  not  now  speaking  of  the  incubation 
of  cultures  for  purposes  of  research  ;  we  are  dealing  with  methods 
of  cultivation  which  are  necessary  for  diagnostic  purposes,  and 
for  these  it  is  usually  sufficient  if  the  temperature  remains  nearly 
constant  at  the  proper  point  for  some  eighteen  hours. 


Fig.  II. 


INCUBATION    OF    CULTURES 


21 


Another  method  I  have  recently  adopted  is  to  use  a  "Thermos  " 
flask,  or,  what  comes  to  the  same  thing,  a  Dewar's  liquid  air 
flask.  This  is  nearly  filled  with  water  at  a  temperature  of  about 
40°  C.  and  the  culture-tube  inserted.  If  a  Thermos  is  used  the 
cap  is  then  applied,  if  a  Dewar's  flask  a  few  drops  of  oil  are 
placed  on  the  surface  of  the  water  (to  prevent  evaporation,  which 
greatly  hastens  the  process  of  cooling).  The  water  and  culture- 
tube  cool  very  gradually,  and  most  pathogenic  organisms  will 
give  an  excellent  growth  before  this  happens.  The  Dewar's 
flask  should  have  a  capacity  of  600  c.c.  and  an  internal  diameter 
at  the  neck  of  i  inch  or  so.  It  can  be  supported  on  a  jam-pot 
with  a  layer  of  cotton-wool  round  the  neck.  I  find  it  cools  at  the 
rate  of  about  half  or  two-thirds  of  a  degree  per  hour. 


Fig.  12. — Dewar's  Flask  adapted  for  Use  as  Incubator. 

a,  Culture  tube ;  b,  vacuum  ;  c,  water  ;  d,  layer  of  oil ;  e,  cotton-wool  round 
mouth  of  jar  for  support. 

The  author  once  succeeded  in  making  a  diagnosis  in  a  case  of 
supposed  diphtheria  by  the  following  method :  The  tube  of 
medium  was  inoculated  from  the  throat,  and  placed  in  a  jam-pot 
which  was  partly  filled  with  water  at  37°  C. ;  a  thermometer  was 
also  inserted,  and  the  whole  placed  near  the  fire.  It  was  watched 
from  time  to  time,  and  moved  near  the  fire  if  the  temperature 
showed  signs  of  falling,  and  vice  vevsa^  until  the  proper  position 
was  found.  Next  morning  there  was  an  excellent  growth,  and 
the  diagnosis  was  made  with  certainty. 

Lastly,  the  author  has  heard  of  a  practitioner  who  was  in  the 


22  CLINICAL    BACTERIOLOGY   AND    HEMATOLOGY 

habit  of  incubating  cultures  at  the  body  temperature  by  carrying 
them  in  an  inner  pocket  during  the  day,  and  taking  them  to  bed 
with  him  at  night  \* 

METHOD  OF  EXAMINING  CULTURES 

Requisites. — i.  Clean  slides  and  cover-glasses.  (These  must 
be  the  thinnest  in  ordinary  use — i.e.^  No.  i.) 

2.  A  platinum  needle  (straight  or  loop). 

3.  A  Bunsen  burner  or  a  spirit-lamp  with  a  tall  flame. 

4.  The  stain  to  be  employed  (see  p.  28). 

5.  Canada  balsam  dissolved  in  xylol.  This  should  be  bought 
ready  for  use. 

6.  A  pair  of  dissecting  forceps. 

7.  Strips  of  white  blotting  or  filter  paper. 

Process. — i.  Sterilize  the  needle,  and  place  a  small  drop  of  water 
(preferably  distilled)  in  the  centre  of  a  clean  slide. 

2.  Take  the  culture-tube  in  the  left  hand  between  the  index  and 
middle  fingers  with  its  mouth  directed  to  the  right  and  (in  the  case 
of  a  culture  on  a  solid  medium)  slightly  downward. 

3.  Burn  the  surface  of  the  plug  in  the  flame.  Remove  the  plug 
with  the  forceps  (previously  sterilized  by  being  passed  slowly 
through  the  flame),  and  place  it  between  the  ring  and  little  fingers 
of  the  left  hand.     Lay  the  forceps  down. 

In  cases  where  you  are  examining  the  culture  for  diagnostic 
purposes  only,  and  do  not  care  if  it  becomes  contaminated  during 
the  process,  it  is  unnecessary  to  take  these  precautions.  The 
cotton-wool  plug  may  then  be  removed  with  the  fingers  and  laid 
down  on  the  table.  As  a  matter  of  fact,  very  few  cultures  do 
become  contaminated,  even  if  no  precautions  are  taken. 

4.  Sterilize  the  needle  in  the  flame  and  allow  it  to  cool. 

5.  Now  introduce  the  needle  into  the  tube,  and  take  up  a  small 
portion  of  the  growth,  taking  care  not  to  scrape  up  the  surface  of 
the  medium  as  you  do  so.  Most  beginners  fall  into  the  mistake 
of  taking  up  far  too  much  of  the  growth,  and  preparing  a  film 
which  is  spread  so  thickly  that  the  individual  bacteria  cannot  be 
distinguished. 

6.  Take  the  plug  up  in  the  forceps,  burn  its  surface  in  the 
flame,  re-plug  the  tube  and  lay  it  down. 

*  Since  the  above  was  written  an  incubator  to  be  used  in  this  way  has  been 
invented. 


I 


METHOD    OF    EXAMINING    CULTURES  23 

7.  Stir  the  droplet  of  water  which  has  been  deposited  on  the  slide 
with  the  tip  of  the  needle,  so  that  the  bacteria  which  it  carries 
are  mixed  with  the  water.  Now  spread  out  the  emulsion  thus 
produced  so  as  to  form  a  patch  about  J  inch  in  diameter.  If  it 
does  not  spread  out  uniformly  it  is  a  sign  that  the  slide  is  not  clean. 

8.  Sterilize  your  needle. 

g.  Allow  the  film  to  dry  spontaneously.  If  you  have  spread  it 
out  sufficiently  this  will  take  a  very  short  time. 

10.  Fix  the  film  by  passing  the  slide  slowly  through  the  flame 
once  or  twice.  This  coagulates  the  albumin  present,  and  the 
bacteria  are  now  fixed  down  so  firmly  that  they  will  not  be  re- 
moved by  subsequent  washing.  The  exact  amount  of  heat  which 
should  be  used  cannot  be  stated,  as  it  varies  according  to  the 
thickness  of  the  slide,  etc.,  and  can  only  be  determined  by  practice. 
It  may  be  estimated  roughly  by  pressing  the  finger  upon  the  upper 
surface  of  the  slide  close  to  the  film,  but  not  touching  it.  The 
slide  should  be  just  uncomfortably  warm  to  the  finger,  but  not  hot 
enough  to  burn  it. 

11.  Filter  a  few  drops  of  the  stain  on  to  the  surface  of  the  film, 
and  allow  it  to  act  for  the  requisite  time.  Exact  details  will  be 
given  in  each  case. 

12.  Next  wash  the  slide  under  the  tap,  blot  it  with  clean  white 
filter-paper,  taking  care  to  avoid  rubbing,  and  warm  it  gently  over 
the  flame  until  absolutely  dry. 

13.  Place  a  drop  of  balsam  on  the  film,  and  apply  a  clean  dry 
cover-glass. 

The  preparation  is  now  ready  for  examination. 
This  also  is  a  process 

which    sounds    more         iniif iiiillrl iiliiiiilli li illllll |||||||pi!l!i^^f 5 ) 

complicated     than     it 

really   is.      The   steps 

are  readily  learnt,  and 

the  whole  process  (ex-  Fig.  13. —Cornet's  Forceps. 

eluding  the  time  spent 

in  staining,  which  may  be  long  or  short)  does  not  take  more  than 

two  or  three  minutes. 

Most  bacteriologists  make  their  films  on  cover-glasses  and  not 
on  slides.  The  steps  are  just  the  same,  except  that  the  cover- 
glasses,  being  much  thinner,  naturally  require  much  less  heating 
to  fix  the  film ;  they  are  passed  rapidly  through  the  flame  three 
times.     It  is  a  great  advantage  to  use  Cornet's  forceps  in  working 


24  CLINICAL    BACTERIOLOGY    AND    HEMATOLOGY 

with  cover-glasses.  These  forceps  are  self-retaining,  and  hold 
the  cover-glass  in  a  horizontal  position,  so  that  stains  can  be 
poured  upon  them  whilst  the  forceps  are  resting  on  the  table. 
In  using  them  put  the  cover-glass  between  the  jaws  of  the 
forceps,  which  must  clip  them  a  little  distance  from  the  corners, 
otherwise  the  stain  will  run  into  the  forceps.  Make  a  point  of 
placing  them  on  the  table  with  the  keyhole  upwards,  so  that  you 
will  always  know  on  which  side  of  the  cover-glass  the  film  has 
been  spread.  But  the  staining  can  be  carried  out  equally  well 
in  a  watch-glass,  and  the  cover-glass  may  be  held  in  dissecting 
forceps. 

It  is  far  easier  and  more  satisfactory  in  every  way  to  make  the 
films  on  the  slides.  Beginners  will  find  that  they  will  break  large 
numbers  of  cover-glasses  (which  must  be  thin),  drop  more  on  the 
floor,  and  will  be  in  constant  doubt  as  to  which  is  the  film  side. 
With  slides  these  difficulties  do  not  occur,  and  the  use  of  forceps 
is  quite  unnecessary. 

It  is  not  absolutely  necessary  to  use  cover-glasses  in  the 
preparation  of  these  bacterial  films  unless  they  are  to  be  kept  for 
future  reference,  and  except  in  such  cases  I  have  discarded  them 
for  years.  To  examine  the  unmounted  film  it  is  only  necessary 
to  dry  it  thoroughly,  and  to  put  the  immersion  oil  (see  p.  27) 
directly  on  to  the  stained  area.  This  method  is  especially  useful 
in  searching  for  the  tubercle  bacillus,  for  no  one  wants  to  keep 
films  of  tuberculous  sputum  after  the  diagnosis  has  been  made, 
and  if  the  necessity  did  arise  a  drop  of  balsam  can  be  placed  on 
the  film  (without  removing  the  oil)  and  the  cover- glass  applied. 
The  one  advantage  of  covering  the  films  at  once  is  that  it  permits 
of  the  use  of  the  low  powers,  and  as  this  should  always  be  done 
by  beginners,  the  covering  of  the  preparations  has  been  recom- 
mended throughout. 

GRAM'S   METHOD   OF   STAINING 

The  method  of  staining  described  above  is  available  for  all 
organisms,  and  therein  consists  its  advantage.  But  other  things 
than  bacteria  are  stained :  pus  cells,  fragments  of  tissue,  debris, 
etc.,  will  all  be  coloured,  and  may  obscure,  or  even  be  mistaken 
for,  bacteria.  Gram's  method  possesses  the  enormous  advantage 
that  by  its  use  the  bacteria  are  coloured,  while  other  structures 
(with  the  exception  of  particles  of  keratin  and  dividing  nuclei) 


I 
I 


gram's  method  of  staining  25 

are  not.  Hence  in  a  film  stained  in  such  a  way  the  bacteria  are 
very  distinct. 

Gram's  method  possesses  another  advantage.  It  is  a  selective 
stain.  Some  bacteria  retain  the  stain,  whilst  others  do  not,  and 
this  fact  is  of  great  value  in  diagnosis.  The  diphtheria  bacillus, 
for  instance,  stains  when  treated  in  the  way  we  shall  describe, 
and  if  an  organism  which  presents  the  character  of  that  bacillus 
does  not  stain  by  the  process  it  must  be  of  some  other  species. 
We  shall  append  a  table  of  the  most  important  pathogenic 
bacteria  which  stain  and  which  do  not  stain  in  this  way. 

Requisites, — i.  AniHne  gentian  violet,  or  carbol  gentian  violet 
(see  p.  30). 

2.  Gram's  iodine  solution  (see  p.  30). 

3.  Absolute  alcohol  or  methylated  spirit. 

Process. — i.  Spread,  dry,  and  fix  the  film  in  the  way  described 
above.  Stain  for  two  or  three  minutes  in  the  aniline  gentian 
violet  or  carbol  gentian  violet. 

2.  Rinse  in  water  to  remove  excess  of  stain  and  flood  with  the 
iodine  solution,  and  allow  the  latter  to  act  for  a  minute. 

3.  Wash  off  the  iodine  solution  with  alcohol,  and  continue  the 
application  of  the  latter  until  no  more  colour  comes  away.  It  is 
best  to  pour  a  little  alcohol  on  the  slide  and  rock  the  latter  from 
side  to  side  for  a  minute  or  so,  then  to  pour  off  the  alcohol  and 
add  a  fresh  supply,  and  continue  this  until  the  alcohol  comes  off 
colourless." 

The  Gram  staining  proper  is  then  finished,  but  if  the  organisms 
present  do  not  stain  by  the  method  the  result  will  be  that  every- 
thing is  decolorized,  and  microscopic  examination  will  reveal 
nothing.  This,  of  course,  might  prove  that  the  organism  did  not 
stain  by  Gram,  but  it  might  also  be  due  to  some  error  in  technique, 
by  which  no  organisms  were  put  on  the  slide  to  begin  with  or  got 
washed  off  at  a  subsequent  stage.  To  exclude  this  possibility 
proceed  to  counterstain  with  some  stain  very  different  in  colour 
from  the  gentian  violet :  dilute  carbol  fuchsin  is  best.  Stain  in 
this  for  a  quarter  of  a  minute — not  more,  for  the  stain  is  a  very 
powerful  one,  and  if  allowed  to  act  too  long  may  displace  the 

*  Some  books  contain  the  absurd  statement  that  the  decolorization  is  to  be 
carried  out  until  the  film  is  a  pale  blue  or  violet  colour.  No  such  rule  can  be 
given.  If  there  is  nothing  that  retains  Gram  the  film  will  be  colourless  when 
finished  ;  if  it  is  a  thick  smear  of  an  organism  retaining  the  stain  it  may  be  3, 
very  deep  violet, 


26  CLINICAL    BACTERIOLOGY   AND    HEMATOLOGY 

stain  from  a  Gram-staining  organism :  wash  for  a  minute  or  so 
under  the  tap,  dry,  and  mount.  Organisms  which  retain  Gram's 
stain  will  be  violet,  almost  black,  organisms  which  do  not  will  be 
red.  Pus,  blood,  etc.,  if  present,  will  also  be  red.  This  counter- 
staining  by  means  of  a  good  contrast  stain  is  really  almost  a 
necessary  sequel  to  Gram's  method,  and  in  practical  diagnostic 
work  the  two  almost  always  go  together. 
Dry  and  mount  as  before. 

The  following  important  bacteria  stain  by  Gram's  method  : 
Staphylococci. 
Streptococci. 
Pneumococci. 
Sarcinae. 

The  skin  coccus  (M.  epidermidis). 
The  bacillus  of  anthrax. 

„  diphtheria. 

„  tetanus. 

„  tuberculosis. 

„  leprosy. 

„  Boas-Oppler. 

The  various  species  of  actinomycosis  fungus  or  streptothrix. 
The  fungi  of  ringworm,  favus,  etc. 
Yeasts. 

The  following  important  pathogenic  bacteria  do  not  stain  by 
Gram's  method  : 
The  gonococcus. 

Diplococctts  intracelhilavis  meningitidis  (Weichselbaum). 
Micrococcus  catavrhalis. 
The  pneumo-bacillus. 
The  Bacilhts  coli  communis. 
The  bacillus  of  glanders. 

„  typhoid  fever. 

„  influenza. 

plague. 
„  soft  sore. 

The  Bacillus  fusiformis. 
The  vibrio  of  Asiatic  cholera. 

The   spirilla   or  spirochsetes  of  relapsing  fever,  syphilis,  and 
Vincent's  angina,  and  all  other  parasitic  protozoa. 


k 


EXAMINATION    OF    FILMS — USE    OF   MICROSCOPE  27 


EXAMINATION  OF  FILMS— USE  OF  MICROSCOPE 

Daylight  is  the  best  illuminant  for  microscopic  work,  and 
the  light  reflected  from  a  white  cloud  opposite  the  sun  is 
best  of  all.  Direct  sunlight  is  useless,  but  the  light  obtained 
from  a  ground -glass  window  on  which  the  sun  is  shining  is 
very  good. 

For  work  at  night  the  light  from  an  incandescent  gas-burner  at 
a  distance  of  2  or  3  feet  is  excellent,  but  an  ordinary  paraffin 
lamp  will  answer  quite  well. 

Having  arranged  for  a  suitable  source  of  light,  turn  the  flat 
mirror  uppermost,  and  move  it  about  until  a  beam  of  light  is 
thrown  on  to  the  condenser.     Remember  : 

In  examining  stained  specimens  use  a  large  diaphragm. 

In  examining  tmstained  objects  use  a  small  diaphragm. 

You  are  now  about  to  examine  a  stained  specimen.  Place  the 
slide  on  the  stage,  putting  the  stained  film  in  the  centre  of  the 
aperture,  and  turn  on  the  low  power.  Look  down  the  eye-piece, 
and  move  the  mirror  about  until  the  field  is  brilliantly  illuminated. 
Focus  the  microscope  (using  the  coarse  adjustment)  until  the 
image  is  clearly  defined.  Now  move  the  slide  about  until  there 
is  a  deeply-stained  area  in  the  centre  of  the  field.  This  area  will 
not  necessarily  be  the  best  for  examination  with  a  higher  power, 
but  it  will  serve  to  catch  the  eye  when  focussing  the  lenses  which 
focus  at  a  short  distance  from  the  object. 

Now  turn  on  the  high  power  (the  \  inch).  Remember  that  the 
*'  working  distance  "  of  all  lenses  is  necessarily  less  than  their 
focal  distance,  and  that  a  ^-inch  lens  focusses  at  a  distance  from 
the  object  which  is  decidedly  less  than  \  inch  ;  so  also  with  the 
other  powers.  Lower  the  lens  until  it  almost  touches  the  object, 
and  screw  up  the  substage  condenser  as  high  as  it  will  go. 
Look  down  the  microscope  and  focus  sHghtly  upwards,  using  the 
coarse  adjustment,  until  you  catch  a  glimpse  of  colour  ;  then 
focus  very  slowly  until  the  object  is  sharply  defined. 

After  a  little  practice  you  will  be  able  to  focus  downwards  on 
to  the  film,  keeping  a  sharp  look-out  for  the  first  appearance  of 
colour,  but  for  beginners  the  foregoing  method  is  easier  and 
safer. 

Study  the  object  with  the  high  power,  and  move  it  about  until 
you  find  an  area  where  the  bacteria  are  neither  too  thickly  nor 


28  CLINICAL    BACTERIOLOGY   AND    HEMATOLOGY 

too  thinly  spread,  and  are  well  stained.  Make  out  as  much  of 
their  appearance  as  you  are  able  to  do  with  this  power.  Very 
much  can  be  done  ;  tubercle  bacilli,  gonococci,  and  many  other 
bacteria,  may  be  recognised  with  this  power,  and  the  peculiar 
arrangement  of  diphtheria  bacilli  can  be  seen. 

Apply  the  clips  to  keep  the  slide  in  place. 

Now  raise  the  tube  of  the  microscope  for  a  short  distance, 
using  the  coarse  adjustment,  and  place  a  small  drop  of  cedar  oil 
on  the  centre  of  the  cover-glass.  Lower  the  tube  (using  the 
coarse  adjustment)  until  the  nozzle  of  the  lens  touches  the  drop 
of  oil ;  then  put  your  head  on  a  level  with  the  stage,  and  continue 
to  focus  downwards,  going  very  carefully,  until  the  lens  almost 
touches  the  cover-glass.  Next  look  down  the  microscope,  and 
focus  upwards,  using  the  fine  adjustment,  until  you  begin  to  see 
colour ;  then  go  more  slowly  until  the  film  is  well  defined. 

Beginners  are  strongly  urged  to  adopt  this  method  of  focussing 
an  oil-immersion  lens  until  they  have  acquired  a  considerable 
amount  of  practice.  It  takes  a  little  time,  but  this  is  well  repaid 
by  the  absence  of  all  danger  or  injury  to  lens  and  cover-glass. 
After  a  time  you  may  lower  the  lens  until  it  touches  the  oil,  and 
then  look  down  the  microscope  and  continue  to  lower  it  with  the 
fine  adjustment. 

After  use,  wipe  the  front  of  the  immersion  lens  with  a  soft  silk 
handkerchief  kept  specially  for  the  purpose,  and  put  the  microscope 
back  into  its  case.  If  oil  or  balsam  should  get  dried  on  the  lens, 
wipe  it  with  a  handkerchief  just  moistened  with  xylol  or  pure 
turpentine,  and  then  wipe  quickly  with  a  dry  handkerchief. 
Never  dip  the  point  of  a  lens  into  xylol  or  alcohol.  Never 
remove  the  front  combination  of  an  oil-immersion  lens  for  clean- 
ing or  any  other  purpose. 

STAINS 

The  following  stains  are  all  that  is  really  necessary  for  the  vast 
majority  of  purposes :  methylene  blue,  basic  fuchsin,  gentian  violet, 
thionin,  and  water-soluble  eosin.  Bismarck  brown  may  also  be 
obtained.  Ten  grammes  of  each  will  last  the  practitioner  for  a 
long  time,  and  this  amount  costs  from  yd.  to  is.  They  should  be 
of  Griibler's  make,  and  can  be  obtained  from  Messrs.  Baker, 
Holborn ;  Baird  and  Tatlock,  Hatton  Garden ;  J.  J.  Griffin, 
Kingsway,  W.C. ;  A.  Fraser,  Teviot  Place,  Edinburgh;  and  from 


STAINS  29 

Messrs.  Southall  or  Philip  Harris,  Birmingham.      Other  firms 
will  also  supply  stains,  but  Griibler's  should  always  be  specified. 

They  are  conveniently  kept  in  a  saturated  solution  of  absolute 
alcohol.     The  following  formulae  are  the  most  useful : 

1.  A  Saturated  Watery  Solution  of  Methylene  Blue. — This  does  not 
keep  very  well,  and  a  fresh  amount  should  be  prepared  after  a 
month.  It  is  mostly  used  for  staining  blood-films,  and  for  staining 
wet  specimens  of  cells  from  the  pleura,  pericardium,  etc. ;  borax 
methylene  blue  will  serve  every  purpose  in  bacteriological  work. 
Instead  of  this  I  now  employ  acid  methylene  blue  (No.  9). 

2.  Loffler^s  methylene  blue  is  prepared  by  adding  30  c.c.  of  a 
saturated  solution  of  methylene  blue  (alcoholic)  to  100  c.c.  of 
a  I  in  10,000  solution  of  caustic  potash. 

The  potash  solution  is  prepared  thus :  Take  i  c.c.  of  a  10  per 
cent,  solution  of  caustic  potash  and  make  up  to  100  c.c.  with 
water ;  shake  thoroughly  and  pour  away  90  c.c. ;  make  up  to 
100  c.c.  with  water,  and  again  shake.  A  sufficiently  close  ap- 
proximation is  made  by  adding  i  minim  of  the  10  per  cent, 
solution  to  2  ounces  of  water. 

This  stain  keeps  fairly  well. 

3.  Hanson's  borax  methylene  blue,  which  I  now  use  in  place  ot 
Loffler's  blue  and  find  much  preferable.  A  stock  solution  (which 
keeps  well)  is  prepared  by  dissolving  2  grammes  of  methylene 
blue  and  5  grammes  of  borax  in  100  c.c.  of  water.  It  is  to  be 
diluted  with  five  to  ten  times  its  volume  of  water  for  use.  It 
may  be  used  whenever  Loffler's  blue  is  recommended.  In 
English  measures  it  is  methylene  blue,  grs.  xxx. ;  borax,  grs.  Ixxv. ; 
aq.,  giii.ss. 

4.  Cavbol  fuchsin  is  made  by  adding  a  saturated  alcoholic  solution 
of  fuchsin  to  carbolic  acid  lotion  (i  in  20)  until  the  fluid  has  lost 
its  transparency,  or  i  part  of  the  saturated  solution  to  9  of  the 
carbolic  acid.     This  keeps  well. 

The  following  is  another  formula  : 

Fuchsin,  i  gramme,  or  grs.  xv. 
I  in  20  carbolic,  100  c.c,  or  giii.ss. 
Glycerin,  50  c.c,  or  3xiv. 

5.  The  above  stain  diluted  with  four  or  five  times  its  volume  of 
water.  Label  "  Dilute  Carbol  Fuchsin."  It  is  usually  prepared 
by  diluting  a  little  strong  carbol  fuchsin  when  required. 


30  CLINICAL    BACTERIOLOGY   AND    HEMATOLOGY 

6.  A  niline  gentian  violet ^  which  is  prepared  as  follows  : 

First  prepare  aniline  oil  water  by  shaking  water  (preferably  dis- 
tilled) with  more  aniline  oil  than  it  will  dissolve ;  a  milky  emulsion 
will  result,  and  this  must  be  allowed  to  settle  for  a  short  time. 
Then  filter  it  through  a  double  thickness  of  filter-paper  which 
has  been  previously  moistened  with  water. 

To  9  parts  of  the  solution  thus  obtained  add  i  part  of  saturated 
solution  of  gentian  violet  in  alcohol. 

This  solution  keeps  badly,  and  it  is  necessary  that  it  should  be 
freshly  prepared,  as  very  important  inferences  are  drawn  from 
results  obtained  with  it.  The  following  keeps  better,  and  answers 
every  purpose. 

Carbolic  Gentian  Violet  (a  substitute  for  aniline  gentian  violet). — 
Add  I  part  of  saturated  alcoholic  solution  of  gentian  violet  to 
9  parts  of  a  I  in  20  carbolic  lotion. 

7.  Carbol  thionin  is  made  by  adding  i  gramme  of  thionin  to 
100  c.c.  of  a  I  in  40  solution  of  carbolic  acid. 

This  stain  keeps  fairly  well,  but  it  must  always  be  filtered 
immediately  before  use,  as  crystals  which  may  have  a  most 
delusive  resemblance  to  long  slender  bacilli  are  frequently  de- 
posited in  it.  A  similar  formation  of  crystals  also  occurs  if  the 
stain  be  allowed  to  dry  on  the  slide. 

In  cold  weather  the  thionin  may  crystallize  nearly  completely  out, 
and  the  fluid  stain  very  badly.  If  kept  in  a  warm  place  for  a  few 
hours  it  will  recover  its  properties,  the  sediment  being  redissolved. 

8.  Eosin  is  used  in  a  4  or  5  per  cent,  watery  solution.  This 
keeps  well.     Red  ink  (slightly  diluted)  will  answer  most  purposes. 

9.  Acid  methylene  blue  is  prepared  by  mixing  2  parts  of  borax 
methylene  blue  (vide  supra) ,  i  part  of  glacial  acetic  acid,  and  7  parts 
of  water.  It  is  only  used  for  staining  cells,  casts,  etc.,  in  wet 
preparations,  and  has  the  advantage  of  dissolving  the  red  blood- 
corpuscles,  which  when  numerous  often  obstruct  the  view  of  the 
more  important  elements.     It  keeps  well. 

Stains  should  be  filtered  before  use.  Where  much  work  is  to 
be  done,  it  is  convenient  to  keep  them  in  bottles  which  are  closed 
with  a  perforated  cork  through  which  a  small  glass  funnel  is 
placed.  A  filter-paper  is  kept  permanently  in  this  funnel,  and  the 
stain  is  filtered  directly  on  to  the  slide  or  cover-glass. 

Gram's  iodine  solution  may  be  mentioned  here,  though  it  is  not  a 
stain.  It  consists  of  a  solution  of  iodine,  i  part ;  iodide  of  potassium, 
2  parts  ;  water,  300  parts.     It  keeps  indefinitely. 


I 


CLEANING    SLIDES   AND    COVER-GLASSES  3I 

CLEANING  SLIDES  AND  COVER-GLASSES 

Slides  and  cover-glasses  must  be  absolutely  clean  when  used  in 
the  bacteriological  laboratory ;  it  is  especially  necessary  that  they 
should  be  free  from  the  slightest  trace  of  grease,  for  this  will 
prevent  fluid  from  spreading  out  into  a  thin  and  uniform  film. 

Slides  are  best  cleansed  by  dropping  them  one  at  a  time  into 
strong  nitric  or  sulphuric  acid,  and  allowing  them  to  soak  for  an 
hour  or  more.  They  are  then  washed  in  running  water  for 
another  hour,  soaked  in  strong  ammonia  for  an  hour,  and  kept  in 
alcohol.  As  methylated  spirit  is  not  so  good  for  this  purpose,  the 
use  of  alcohol  is  rather  expensive ;  but  it  is  not  absolutely 
necessary,  and  the  slides  may  be  stored  in  a  solution  of  ammonia 
(about  I  in  10)  until  required.  When  about  to  be  used,  they  are 
to  be  wiped  dry  with  an  old  linen  handkerchief  kept  specially  for 
the  purpose.  This  handkerchief  should  be  as  old  as  possible,  and 
should  have  been  washed  until  it  has  begun  to  fall  to  pieces. 

Another  and  more  rapid  method  is  to  place  the  slides  in  a  thin 
glass  or  earthenware  vessel  and  moisten  them  with  methylated 
spirit,  and  then  to  cover  them  completely  with  strong  commercial 
nitric  acid,  placing  the  vessel  in  the  open  air.  In  a  little  while 
the  acid  will  become  very  hot  and  emit  copious  fumes.  When 
the  ebullition  has  ceased,  any  fat  which  may  be  present  will  have 
melted,  and  will  form  a  pellicle  on  the  surface,  whilst  other  organic 
materials  will  have  been  destroyed.  The  acid  is  then  to  be  poured 
off,  taking  care  to  remove  the  pellicle  of  grease  with  it,  and  a 
stream  of  water  allowed  to  fall  into  the  vessel  until  the  acid  has 
been  washed  away.  The  slides  (and  the  method  is  a  good  one  for 
cover-glasses  also)  may  then  be  placed  in  absolute  alcohol  or 
methylated  spirit,  and  only  require  drying  to  be  ready  for  use. 

Another  method  which  I  have  recently  used  and  found  excellent 
is  to  boil  them  in  a  5  per  cent,  solution  of  lysol ;  they  may  be 
allowed  to  remain  in  this  fluid  until  required,  and  are  then 
polished  with  a  handkerchief — and  it  may  be  pointed  out  that 
thorough  friction  is  a  sine  qua  non  whatever  method  of  cleaning  be 
adopted.  Slides  and  cover-glasses  after  use  may  be  soaked  in  the 
lysol  solution,  a  pot  of  which  should  be  kept  at  hand  for  the 
purpose ;  they  will  be  sterilized  and  partially  cleaned  in  a  few 
hours. 

When  no  properly-cleaned  slides  are  at  hand,  the  following 
method  may  be  adopted,  though  it  is  not  so  good :  Dip  the  end 


32  CLINICAL    BACTERIOLOGY   AND    H.EMATOLOGY 

of  a  clean  handkerchief  in  strong  spirit  (absolute  or  rectified)  and 
wipe  the  slide  with  it,  using  a  considerable  amount  of  friction. 
Now  dry  it  with  the  special  handkerchief  mentioned  above,  heat 
it  thoroughly  in  a  smokeless  flame,  and  allow  to  cool  completely. 
Spread  the  film  on  the  surface  which  was  exposed  directly  to  the 
flame. 

Cover-glasses  are  cleaned  in  the  method  advised  for  slides,  and 
should  be  stored  in  strong  alcohol  smelling  strongly  of  ammonia. 
They  are  wiped  with  the  special  handkerchief  immediately  before 
use. 

When  cover-glasses  are  to  be  used  for  covering  films  spread 
upon  slides  (as  is  generally  the  case  if  the  method  recommended 
in  this  book  is  adopted)  it  is  quite  sufficient  to  wipe  them  carefully 
with  a  clean  handkerchief  moistened  with  spirit,  and  then  to  dry 
them. 

After  slides  or  cover-glasses  have  been  cleaned,  the  utmost  care 
must  be  taken  that  they  do  not  come  in  contact  with  the  skin,  or 
a  thin  film  of  grease  will  be  deposited  upon  them. 


PIPETTES 

Glass  pipettes  for  the  collection  of  pathological  fluid  for 
bacteriological  examination  are  very  frequently  required.  They  are 
readily  made  from  a  piece  of  quill  glass  tubing,  and  a  few  should 
always  be  kept  in  stock  against  emergencies. 

One  form  consists  of  a  bulb  about  J  inch  long,  each  end  of 
which  is  drawn  out  into  a  narrow  tube  at  least  6  inches  long, 
tapering  gradually  to  the  extremities  (Fig.  14,  a).  To  make  such 
a  pipette,  take  a  piece  of  glass  tubing  about  6  inches  long  and  a 
J  inch  wide,  and  heat  it  in  a  luminous  gas  flame  at  a  point  J  inch 
or  so  from  the  centre.  Continue  the  heat  until  the  glass  is 
thoroughly  softened  over  at  least  J  inch  of  its  length,  turning 
the  tube  round  all  the  time  ;  then  remove  it  from  the  flame 
and  draw  the  two  ends  apart  with  a  steady,  uniform  pull,  so 
that  the  heated  portion  draws  out  into  a  capillary  tube  several 
inches  in  length.  Repeat  the  process  at  a  point  about  -J  inch 
from  the  tapering  end  of  the  larger  portion  of  the  tube ;  heat  the 
bulb,  and  then  seal  off  both  ends  of  the  capillary  portion  before 
the  bulb  cools. 

The  pipettes  are  necessarily  sterile,  having  been  drawn  out  of 
partially  melted  glass,  and  they  will  remain  sterile  indefinitely. 


PIPETTES  33 

Of  course,  the  exterior  of  the  glass  will  become  contaminated, 
and  it  should  be  passed  through  the  flame  before  use. 

The  ends  of  the  tube  being  sealed  up  while  the  bulb  contains 
heated  air,  it  follows  that  the  bulb  will  contain  a  partial  vacuum 
on  cooling.  This  fact  is  made  use  of  in  the  collection  of 
specimens.  Suppose,  for  instance,  we  wish  to  take  some  blood 
from  a  heart  at  a  post-mortem  examination  for  investigation  at  a 
distance.  A  point  on  the  surface  of  the  heart  is  first  seared  with  a 
hot  iron  to  destroy  any  germs  which  might  be  present,  and  the 
end  of  the  pipette  (still  sealed)  is  thrust  through  into  one  of  the 
cavities.  It  is  then  broken  off  by  dexterous  pressure  against  the 
heart  wall,  and  the  pipette  will  fill  slowly  with  the  blood.    Another 


D 


c 
Fig.  14. — Pipettes. 

method  is  to  break  off  the  tip  of  the  pipette  and  to  warm  the 
bulb  before  making  the  puncture.  The  fluid  will  rise  as  the 
bulb  cools ;  or  both  ends  may  be  broken  up  and  the  fluid  drawn 
into  the  bulb  by  gentle  suction. 

Under  any  circumstances  both  ends  of  the  pipette  must  be  sealed 
up  in  a  flame  (the  flame  of  a  wax  match  will  answer  at  a  pinch), 
and  the  tube  labelled. 

Another  variety  of  pipette  which  was  much  used  for  the  collec- 
tion of  blood  for  the  purpose  of  testing  the  serum  reaction  for 
typhoid  fever,  is  drawn  out  to  a  point  at  one  end  only,  the  other 
being  left  wide  and  separated  from  the  bulb  by  a  constriction 
(Fig.  14,  b).  The  open  end  should  be  loosely  plugged  with 
cotton-wool,  and  serves  as  a  mouthpiece.  The  manufacture  of 
these  pipettes  presents  a  little  difficulty,  but  a  small  amount  of 
practice  will  enable  the  practitioner  to  turn  out  a  perfectly 
serviceable  one  on  occasion. 

Wright's  blood  capsules  (Fig.  15)  are  the  best  contrivances  for 
collecting  blood  when  the  serum  has  to  be  examined,  and  have 
quite  replaced  the  pipettes  described  above.     They  can  be  pre- 

3 


34 


CLINICAL    BACTERIOLOGY   AND    H.EMATOLOGY 


pared   easily   after   a   little    practice,   or   may   be   bought    from 
R.  B.  Turner,  ii,  Foster  Lane,  E.G.,  at  a  moderate  price. 

A  Wright's  blood  capsule  consists  of  a  piece 
of  glass  tubing  which  is  drawn  out  straight  to  a 
narrow  point  at  one  end,  whilst  the  other,  also 
drawn  out  to  a  point,  is  curved  round  parallel 
with  the  main  tube  in  the  shape  of  a  U  (Fig.  15). 
To  use  it,  proceed  as  follows  :  Prick  the  patient's 
ear,  or,  if  you  prefer  it,  the  finger  (after  the 
application  of  a  wide  indiarubber  band),  and 
squeeze  out  a  large  drop  of  blood.  Place  the 
tip  of  the  curved  end  of  the  pipette  in  this  drop, 
holding  the  pipette  with  the  straight  end  point- 
ing upward  (Fig.  16),  and  you  will  find  that  the 
blood  will  run  rapidly  into  the  curved  tube  by 
capillary  attraction ;  continue  to  squeeze  out 
more  blood  and  to  suck  it  up  until  you  have 
collected  as  much  as  you  want.  It  should  be 
quite  easy  to  get  the  pipette  half  full.  Then  gently  warm  the 
tapering  portion  of  the  straight  end  of  the  pipette  in  a  spirit-lamp 
or  match-flame,  and  afterwards  seal  the  tip  (Fig.  17).     As  the  air 


Fig.  15.     (About 
§  full  size. ) 


which  is  now  imprisoned  in  the  upper  part  of  the  pipette  contracts 
it  will  suck  the  blood  from  the  curved  limb  into  the  body  of  the 
pipette,  which  can  be  inverted  and  the  blood  shaken  into  the  tip, 
but  take  care  not  to  do  this  until  the  glass  is  coldj  as  the  serum  loses 
some  of  its  properties  when  heated.      If  you  are  not  going  to 


PIPETTES 


35 


examine  the  serum  for  some  time  seal  the  other  end  to  prevent 
evaporation. 

When  the  blood  has  coagulated  it  will  begin  to  contract  away 
from  the  sides  of  the  tube,  the  serum  being  forced  out  of  the  clot. 
When   this   process  is  complete   there  will   be  a  central  dark- 


#^c^ 


Fig.  17. 


Fig.  18. 
A,  Before  coagulation  :  B,  after,  showing  clot  suspended  in  clear  serum. 

coloured  clot  suspended  in  clear  serum.  The  tube  can  now  be 
notched  with  a  file  and  broken,  and  the  serum  removed  with  a 
pipette  and  used  for  Widal's  reaction,  the  estimation  of  the 
opsonic  index,  etc.  If  much  serum  is  required,  the  pipette  can 
be  hung  by  its  crook  (straight  end  downward)  in  the  bucket  of  a 

3—2 


36  CLINICAL    BACTERIOLOGY    AND    H^MATOLOGi^ 

centrifugal  machine  and  centrifugalized.     In  this  way  the  clot  is 
driven  to  the  bottom  and  a  large  crop  of  serum  obtained. 

Since  some  practitioners  seem  to  have  difficulty  in  collecting 
the  quantity  of  blood  desirable  for  the  opsonic  and  other  tests,  a 
few  more  notes  on  the  process  may  be  added.  I  personally  prefer 
to  obtain  the  blood  from  the  ear,  and  proceed  as  follows  :  the 
lobe  is  well  rubbed^by  means  of  a  piece  of  lint,  until  it  is  markedly 
hyperaemic,  and  is  then  punctured  on  its  lower  border  with  a 
Hagedorn's  needle  or  platino-iridium  hypodermic  needle  (previously 
sterilized  in  the  flame).  This  is  done  by  a  short,  sharp  "jab," 
and  should  not  be  felt  as  pain  at  all ;  I  have  frequently  done  it 
without  waking  a  sleeping  child.  The  needle  is  then  laid  down, 
and  if  the  blood  flows  out  in  large  drops  (as  usually  happens 
if  the  ear  has  been  well  rubbed)  it  is  collected  without  the 
slightest  difficulty,  and  this  is  specially  likely  to  be  the  case  if 
you  puncture  the  ear  of  the  side  on  which  a  patient  has  been 
lying.  If  it  does  not  flow  readily,  take  the  lobe  of  the  ear 
between  the  forefinger  and  thumb  of  both  hands,  and  squeeze  it 
gently  so  that  all  the  blood  in  the  lobe  is  forced  through  the 
puncture,  and  collect  the  drop  thus  formed.  When  this  has 
been  done  the  lobe  of  the  ear  will  be  full  of  blood  again,  and  a 
second  drop  can  be  milked  out.  Repeat  this  process  as  often  as 
necessary.  There  are  few  patients  from  whom  several  pipettes 
half  full  cannot  be  collected  from  a  single  slight  puncture. 

Many  pathologists  prefer  the  finger.  In  that  case  an  excellent 
needle  can  be  made  by  softening  a  small  piece  of  capillary  tubing 
in  the  flame  and  separating  the  two  ends  rapidly.  This  will  give 
two  tapering  tubes,  which  can  be  broken  off  so  as  to  leave  a  very 
sharp  point,  with  which  the  skin  at  the  side  of  the  nail  can  be 
punctured  almost  painlessly.  The  ligature  is  then  applied  as  in 
the  figure,  and  the  patient  bends  his  finger  forcibly,  squeezing 
out  two  or  three  drops  of  blood.  After  these  have  been  collected 
the  bandage  must  be  removed  and  the  hand  allowed  to  hang 
down,  so  that  the  finger  refills  with  blood.  It  is  rebandaged  and 
rebent,  and  more  blood  obtained.  This  process  should  be  learnt, 
since  it  is  the  simplest  one  by  which  the  practitioner  can  obtain 
blood  from  himself,  as  is  often  necessary  in  opsonic  work. 


SECTION  II 
DIAGNOSIS  OF  CERTAIN  DISEASES 

DIPHTHERIA 

Diphtheria  is  a  local  disease  with  general  symptoms.  The  local 
symptoms,  are  due  to  the  local  action  of  the  bacillus  which  causes 
the  disease,  while  the  general  symptoms  are  due  to  the  toxin  or 
poison  which  they  produce,  and  which  is  carried  in  the  blood- 
stream to  the  brain,  heart,  and  other  organs.  Now,  the  local 
symptoms  are  comparatively  unimportant,  and  it  is  to  the  general 
symptoms  caused  by  the  toxin  that  diphtheria  owes  the  greater 
part  of  its  high  mortality.  Diphtheria  antitoxin  neutralizes  this 
toxin  (much  in  the  same  way  as  an  alkali  neutralizes  an  acid), 
and  prevents  it  from  harming  the  vital  structures ;  but  it  does  not 
repair  the  harm  that  the  toxin  has  done.  It  is  obvious,  therefore, 
that  we  must  not  make  our  diagnosis  of  diphtheria  from  the 
general  symptoms  if  the  antitoxin  treatment  is  to  do  any  good. 
The  diagnosis  is  to  be  made  from  the  local  symptoms,  and  this  is 
what  we  can  rarely  do  by  ordinary  clinical  methods  at  a  stage 
sufficiently  early  to  get  the  full  value  of  the  antitoxin  treatment. 

The  practitioner  has  a  choice  of  two  methods.  He  may  inject 
all  patients  who  suffer  from  sore  throats  which  present  the  slightest 
resemblance  to  those  seen  in  diphtheria,  or  he  may  employ 
bacteriological  methods  of  diagnosis.  The  former  method  may  be 
applicable  in  an  epidemic  of  diphtheria,  but  suspicious  throats  are 
common  and  antitoxin  expensive.  In  most  cases  it  is  necessary 
to  have  recourse  to  the  second  method. 

Most  sanitary  authorities  have  now  recognised  that  it  is  their 
duty  and  privilege  to  provide  for  the  bacterial  investigation  of 
supposed  diphtheria  free  of  charge  to  doctor  and  patient,  and 
supply  outfits  to  be  used  for  taking  the  material  and  transmitting 
it  to  the  laboratory.      When  the  practitioner  lives  within  easy 

57 


38  CLINICAL    BACTERIOLOGY   AND    H.EMATOLOGY 

reach  of  the  laboratory  (so  that  the  swabs  may  reach  it  quickly) 
it  is  his  bounden  duty  to  avail  himself  of  the  opportunity  thus 
afforded  of  getting  a  free  opinion  from  a  specialist. 

But  the  case  of  a  practitioner  living  at  a  distance  is  somewhat 
different.  Suppose  the  patient  is  seen  on  the  first  day  of  the 
illness,  an  unusual  occurrence  except  in  an  epidemic.  The  swab 
is  taken,  despatched  by  post,  and  reaches  the  laboratory  on  the 
second  day.  It  is  inoculated,  and  the  culture  is  incubated  and 
examined  on  the  third  day,  the  result  reaching  the  practitioner 
about  noon  on  that  day.  Now,  the  mortality  of  diphtheria  which 
is  treated  with  antitoxin  on  the  first  day  is  very  small — certainly 
less  than  5  per  cent. — while  the  mortality  in  cases  in  which  its 
use  is  not  commenced  until  the  third  day  is  much  higher — probably 
from  10  to  15  per  cent.,  or  even  higher.  In  other  words,  from 
five  to  ten  patients  out  of  every  hundred  lose  their  lives  if  the 
doctor  waits  for  the  result  of  the  bacteriological  examination. 
It  is  therefore  highly  advisable  that  every  practitioner  should 
provide  himself  with  a  bacteriological  microscope,  and  should  at 
least  examine  a  film  prepared  directly  from  the  swab  and  stained 
in  the  manner  described  below.  He  should  also  make  cultures  or 
send  a  swab  to  the  laboratory  for  examination. 

Swabs  and  outfits  are  provided  by  the  laboratory  where  the 
^examination  is  made,  or  can  be  bought  from  most  manufacturing 
chemists  and  instrument-makers.  A  swab  consists  of  a  steel  or 
copper  (aluminium  would  be  better)  wire,  the  extremity  of  which 
is  covered  drumstick  fashion  with  a  tightly-fitting  roll  of  cotton- 
wool. The  other  end  is  pushed  through  a  cork,  and  the  whole  is 
contained  in  a  stout  glass  tube.  It  is  sterilized  before  use.  These 
swabs  may  be  readily  made  at  home.  A  test-tube  is  fitted  with 
a  good  cork  through  which  is  passed  a  stout  steel  knitting-needle. 
This  should  be  long  enough  to  pass  nearly  to  the  bottom  of  the 
tube  when  the  cork  is  in  place,  and  the  end  which  is  to  be 
outside  the  tube  should  be  cut  off  short.  The  other  must  be 
roughened  by  a  few  strokes  of  a  file.  A  small  piece  of  cotton- 
wool (unmedicated)  is  then  held  between  the  thumb  and  finger  of 
the  left  hand,  transfixed  with  the  roughened  end  of  the  wire,  and 
twisted  round  it.  The  swab  is  now  placed  loosely  in  the  tube  and 
sterilized  by  dry  heat  (see  ante,  p.  7).  It  is  allowed  to  cool  in  the 
sterilizer,  and  the  cork  is  pushed  home  into  the  tube  as  soon  as  it 
is  cold  enough  to  handle.  These  swabs  will  keep  indefinitely, 
and  a  stock  of  them  should  always  be  kept  at  hand,  as  they  are 


DIPHTHERIA  39 

very  handy  for  other  purposes.  After  use  the  cotton-wool  should 
be  burnt  off  in  a  Bunsen  burner  or  spirit-lamp,  and  another 
piece  appHed  and  the  whole  resterilized. 

If  a  practitioner  should  see  a  supposed  case  of  diphtheria  when 
he  is  unprovided  with  a  swab  he  can  readily  extemporize  one 
which  will  answer  sufficiently  well  out  of  some  cotton-wool  (non- 
medicated),  a  wooden  skewer  or  pen-holder,  and  a  glass  phial. 
The  wool  is  wrapped  round  the  tip  of  the  skewer,  and  (after  the 
swab  has  been  taken)  the  latter  is  placed  in  the  phial  in  such  a 
position  that  the  cotton-wool  does  not  touch  the  glass ;  the  place 
between  the  skewer  and  the  neck  of  the  bottle  is  plugged  with 
cotton-wool.  It  is  not  absolutely  necessary  to  sterilize  the  swabj 
although  it  is  a  very  great  advantage  to  do  so  if  time  permits. 

The  method  of  taking  the  swab  is  of  great  importance,  and  must 
be  carried  out  in  full  detail.  It  is  necessary  that  the  patient 
should  not  have  had  an  antiseptic  gargle  or  application  for  at 
least  two  hours  previously.  It  is  also  advisable  to  allow  him  to 
drink  some  beef-tea  or  boiled  water  {not  milk,  for  this  may  contain 
certain  bacilli  which  closely  resemble  those  of  diphtheria)  imme- 
diately before  the  process.     This  will  serve  to  cleanse  the  parts. 

Requisites. — i.  A  good  light. 

2.  The  swab  in  its  tube. 

3.  A  tongue  depressor.  The  form  which  is  hinged  so  as  to 
bend  at  a  right  angle  is  most  convenient. 

4.  A  vessel  containing  antiseptic  lotion  or  boiling  water. 
Method. — I.  Place  the  patient  so  as  to  face  the  light.      If  a 

small  child,  he  should  be  held  on  his  nurse's  lap,  with  a  blanket 
wrapped  round  his  chest  so  as  to  restrain  his  arms. 

2.  Loosen  the  cork  in  the  tube  so  that  the  swab  may  be  with- 
drawn with  one  hand,  and  place  it  at  a  convenient  spot  on  your 
right  side. 

3.  Get  the  patient  to  open  his  mouth,  insert  the  tongue 
depressor  (held  in  the  left  hand),  and  get  a  clear  view  of  the  area 
chiefly  affected.  Do  not  proceed  with  the  process  (if  it  can  be 
avoided)  until  you  have  done  this. 

4.  Take  the  cork  between  the  finger  and  thumb  of  the  right 
hand  and  pass  the  swab  into  the  patient's  mouth,  taking  great 
care  not  to  touch  his  lips,  tongue,  or  palate.  Press  it  firmly 
against  the  area  which  you  wish  to  examine,  and  rotate  it  between 
the  finger  and  thumb  so  as  to  remove  some  of  the  secretion,  and, 
if  possible,  some  of  the  membrane.     Withdraw  the  swab,  again 


40  CLINICAL    BACTERIOLOGY   AND    H.EMATOLOGY 

taking  care  not  to  touch  any  part  of  the  mouth,  and  replace  it  in 
the  tube. 

5.  Withdraw  the  tongue  depressor,  and  place  it  in  the  anti- 
septic lotion  or  boiling  water. 

6.  Push  the  cork  home  into  the  tube. 

Method  of  examining  the  Swab. — This  may  be  carried  out  by  means 
of  stained  films,  prepared  directly  from  the  swabs,  or  by  means 
of  cultures.  The  former  method  is  less  useful  than  the  latter, 
but  we  shall  consider  it  first,  as  it  can  be  performed  by  anyone 
who  possesses  a  microscope  carrying  a  yV^nch  oil-immersion 
lens,  and  often  gives  valuable  information.  Moreover,  it  does 
not  take  long,  and  but  little  delay  is  caused. 

Requisites. — i.  Clean  slides  and  cover-glasses. 

2.  Stains. — Loffler's  blue  or  carbol  thionin,  and  aniline  gentian 
violet. 

3.  Gram's  iodine  solution  and  alcohol — methylated  spirit  will  do. 

4.  Strips  of  white  filter  or  blotting  paper. 

5.  Balsam. 

Method. — Prepare  a  film  in  the  following  way :  Rub  the  swab 
on  the  middle  of  a  clean  slide,  so  as  to  spread  some  of  the  secretion 
into  a  thin  layer  on  the  surface.  Allow  it  to  dry,  and  fix  it  by 
passing  it  slowly  through  the  flame,  until  the  upper  surface  is  just 
too  hot  to  prevent  your  pressing  your  finger  upon  it  in  comfort. 
Allow  it  to  cool. 

Now  filter  a  few  drops  of  Loffler's  blue  or  carbol  thionin  on 
to  the  film,  and  allow  it  to  act  for  two  minutes.  Wash  under 
the  tap. 

Dry  by  pressing  carefully  with  strips  of  blotting-paper,  and 
then  in  the  flame.  Place  a  drop  of  balsam  upon  the  film,  and 
apply  a  cover-glass. 

Prepare  a  second  film,  and  stain  by  Gram's  method  (p.  24), 
counterstaining  by  dilute  carbol  fuchsin. 

The  films  are  now  examined  microscopically  (see  p.  27).  We 
shall  defer  the  description  of  the  points  upon  which  a  diagnosis 
is  to  be  based  until  we  deal  with  the  examination  of  cultures. 

Cultural  Methods. 

The  diphtheria  bacillus  grows  best  at  or  near  the  body 
temperature  (about  37°  C),  and  flourishes  on  almost  all  culture 
media.    But  agar  is  scarcely  ever  used  in  growing  it  for  diagnostic 


DIPHTHERIA  4I 

purposes ;  this  medium  serves  well  for  the  cultivation  of  a  great 
many  organisms,  some  of  which  are  almost  always  present  in  the 
mouth,  so  that  cultures  made  upon  it  are  usually  very  impure. 
We  use  a  medium  which  permits  the  development  of  the  diphtheria 
bacillus,  and  inhibits  that  of  most  other  organisms.  The  best  is 
solidified  blood-serum.  ^ 

The  method  in  which  the  medium  is  inoculated  is  as  follows : 
The  tube  of  culture  medium,  and  the  tube  containing  the  swab, 
are  held  side  by  side  between  the  index  and  middle  fingers  of  the 
left  hand,  the  mouths  of  the  tubes  pointing  to  the  right  and 
slightly  downwards.  The  plug  of  the  culture-tube  is  then  singed, 
removed  by  means  of  a  pair  of  forceps,  and  placed  between  the 
ring  and  little  fingers  of  the  left  hand.  The  cork  and  wire  of  the 
swab-tube  are  now  withdrawn,  and  the  cotton-wool  plug  is  inserted 
into  the  culture-tube,  and  passed  onwards  until  it  reaches  the 
sloped  surface  of  the  medium.  It  is  then  rubbed  gently  on  the 
latter,  and  twisted  round  and  round  so  that  every  part  of  the  swab 
may  come  into  contact  with  the  medium.  If  there  is  a  piece  of 
membrane  special  care  should  be  taken  to  see  that  it  is  rubbed 
on  the  surface,  for  it  is  here  that  we  are  most  likely  to  find 
the  bacilli.  The  swab  is  now  withdrawn  and  replaced  in  the 
tube,  and  the  cotton-wool  plug  of  the  culture-tube  singed  and 
replaced. 

The  tube  thus  inoculated  must  now  be  incubated  for  about 
eighteen  hours  at  a  temperature  not  exceeding  37°  C,  and  is  then 
ready  for  examination. 

Hewlett  has  suggested  a  useful  method  which  may  be  carried 
out  without  any  special  apparatus,  the  white  of  a  hard-boiled  egg 
being  used  as  the  culture  medium.  Take  a  fresh  egg  and  boil  it 
for  ten  minutes  or  more,  and  allow  it  to  cool.  Now  take  a 
narrow-mouthed  wineglass  (or  a  wide-mouthed  bottle,  which  is 
better),  and  rinse  it  out  with  perchloride  of  mercury  lotion. 
Sterilize  a  knife  by  passing  it  slowly  through  the  flame,  and  cut 
off  the  top  of  the  egg,  care  being  taken  not  to  cut  into  the  yolk. 
Invert  the  egg  into  the  wineglass  (which  must  be  narrow  enough 
to  prevent  the  egg  from  dropping  down  into  it),  taking  care  that 
none  of  the  lotion  touches  the  cut  surface.  This  is  the  culture 
medium,  and  it  is  sterilized  ready  for  inoculation.  At  a  pinch 
it  may  be  incubated  in  a  warm  corner  near  the  fire,  near  the  hot- 
water  cistern,  or  other  warm  place. 


42  CLINICAL    BACTERIOLOGY   AND    H^EMATOLOGY 

Examination  of  the  Cultures. 

1.  Naked-eye. — Each  living  diphtheria  bacillus  which  has  been 
deposited  upon  the  surface  of  the  culture  medium  and  kept  at  a 
suitable  temperature  will  develop  into  a  colony  of  bacilli ;  and 
these  colonies  are  fairly  distinctive,  being  different  from  those 
which  are  formed  by  most  other  organisms.  The  expert 
bacteriologist  can  often  give  an  accurate  guess  as  to  the  presence 
or  absence  of  diphtheria  bacilli  by  mere  inspection  of  the 
cultures.  The  colonies  formed  by  diphtheria  bacilli  on  solidified 
blood-serum  are  small  round  raised  spots ;  they  are  variable  in 
size,  but  rarely  exceed  that  of  the  head  of  a  medium-sized  pin. 
They  are  white  or  grey  in  colour,  and  opaque.  They  do  not 
tend  to  run  together  so  as  to  form  a  uniform  film  over  the 
surface  of  the  medium,  but  remain  discrete  even  when  closely 
packed.  Some  cocci  form  colonies  which  closely  resemble  those 
of  diphtheria,  but  they  rarely  become  elevated  so  high  above  the 
surface  in  the  same  space  of  time. 

2.  Microscopical. — Prepare  films  by  the  method  described  on 
p.  22  following  out  all  steps  in  the  fullest  detail.  Stain  one  of 
them  (step  ii)  with  Lolfler's  blue  or  carbol  thionin,  allowing  the 
stain  to  act  for  two  minutes,  and  the  other  by  Gram's  method. 

In  removing  some  of  the  growth  to  make  the  film,  remember 
the  facts  just  stated  as  to  the  characters  of  the  colonies  of  the 
bacillus,  and  select  a  colony  presenting  those  characters  (especially 
that  of  elevation),  if  one  is  present.  If  there  is  no  apparent 
growth  in  the  tube  take  "  sweeps  "  of  the  whole  surface.  This  is 
conveniently  done  by  means  of  a  platinum  loop  shaped  like  a 
stirrup,  the  flat  bar  being  drawn  along  the  surface  of  the  medium 
from  bottom  to  top,  just  as  a  rake  is  drawn  along  a  flower-bed. 

Now  examine  your  specimens  in  the  way  described  on  p.  27. 

Characters  of  the  Diphtheria  Bacillus. 

The  following  are  the  chief  points  which  are  considered  in 
deciding  whether  a  given  stained  slide  does  or  does  not  show  the 
diphtheria  bacillus. 

I.  The  shape  of  the  bacillus  is  very  variable,  and  this  is  a 
feature  which  often  affords  us  great  assistance ;  a  specimen  in 
which  all  the  bacilli  present  resemble  each  other  exactly  in  shape 
and  size  is  not  from  a  case  of  diphtheria.     Diphtheria  bacilli  are 


DIPHTHERIA  43 

narrow  rods ;  they  are  either  straight  or  slightly  curved  in  an  arc 
of  a  large  circle  or  into  an  /  shape  (Plate  I.,  Figs,  i  and  2). 
Their  ends  are  usually  rounded,  but  it  is  not  uncommon  to  find 
forms  with  one  end  or  both  sharply  pointed.  Lastly,  clubbed 
forms  are  to  be  met  with  in  almost  all  cultures,  though  they  are 
most  frequent  in  those  which  have  been  incubated  for  several 
days ;  they  may  be  compared  to  a  note  of  exclamation  (!). 

2.  Size. — Two  well-marked  varieties  occur.  The  long  form 
is  about  as  long  as  a  tubercle  bacillus  (to  compare  it  with 
an  organism  with  which  the  practitioner  may  readily  become 
acquainted),  or  somewhat  longer ;  it  is  decidedly  thicker.  The 
short  form  is  only  about  half  as  long  and  thick  in  proportion. 

We  do  not  know  anything  as  to  the  difference  in  pathogenicity 
(if  any)  of  the  long  and  the  short  varieties  of  the  diphtheria 
bacillus.  They  appear  to  "breed  true"  for  long  periods,  and 
cases  of  diphtheria  caused  by  the  one  appear  to  have  as  high  a 
mortality  as  those  caused  by  the  other. 

Hoffmann's  bacillus  is  dealt  with  subsequently. 

3.  Staining  Reactions. — The  diphtheria  bacillus  stains  readily 
with  all  the  stains  in  common  use  for  bacteriological  purposes. 
It  usually  (but  not  invariably)  stains  irregularly,  deeply-stained 
portions  alternating  with  others  which  remain  colourless.  This 
gives  rise  to  a  beaded  appearance,  and  forms  sometimes  occur 
which  can  hardly  be  distinguished  from  short  chains  of  strep- 
tococci. When  a  powerful  stain  is  applied  for  a  long  time  this 
appearance  may  be  lost. 

The  diphtheria  bacillus  stains  deeply  with  thionin.  This  often 
affords  a  certain  amount  of  help  in  the  diagnosis,  as  many  other 
bacilli  do  not  stain  nearly  so  deeply  in  the  same  time. 

It  stains  by  Gram's  method.  A  beginner  should  always  test 
his  results  in  this  way :  If  suspicious  bacilli  do  not  retain  the 
violet  stain  they  are  not  those  of  diphtheria. 

4.  Arrangement. — This  is  a  most  characteristic  feature,  but 
it  is  one  which  is  difficult  to  describe.  The  old  comparison  to 
the  strokes  which  form  a  Chinese  letter  is  a  fairly  good  one; 
the  bacilli  lie  in  little  groups,  some  lying  parallel  to  one  another, 
and  some  at  various  angles  with  these.  The  characteristic 
arrangement  is  best  seen  in  a  specimen  made  from  a  pure 
culture  of  the  short  form. 

Before  coming  to  a  conclusion  as  to  the  presence  or  absence  of 
diphtheria  bacilli  from  an  examination  of  a  stained  film,  make  a 


44  CLINICAL    BACTERIOLOGY   AND    HEMATOLOGY 

very  thorough  search ;  if  no  bacilli  are  seen,  make  several  more 
films  and  examine  them.  When  you  see  a  group  of  bacilli, 
examine  it  carefully,  noting  each  characteristic  and  comparing 
it  with  those  described  above. 

The  beginner  is  strongly  recommended  to  procure  a  series  of 
slides  of  diphtheria  bacilli  from  a  bacteriological  laboratory,  and 
to  study  them  carefully. 

Hoffmann's  bacillus  (Plate  I.,  Fig,  2)  is  probably  a  modified 
form  of  the  true  diphtheria  bacillus,  and  is  frequently  met  with 
in  throat  cultures.  It  is  about  as  long  as  the  short  form  of 
diphtheria  bacillus,  but  decidedly  plumper,  and  is  more  uniform 
in  shape  and  size.  These  bacilli  stain  uniformly  and  deeply. 
They  exhibit  the  same  arrangement,  and  are  often  grouped  in 
pairs.     Clubbed  forms  do  not  occur. 

The  diphtheria  bacilli  which  occur  in  films  made  direct  fvom 
the  swab  are  similar  to  those  seen  in  cultures,  but  are  often 
thicker ;  they  may  stain  uniformly,  and  clubbed  forms  are  rare. 
It  is  unusual  to  be  able  to  make  an  absolute  diagnosis  as  to  their 
nature,  but  it  may  be  done  at  times.  Yet  such  an  examination  is 
often  useful.  If  suspicious  bacilli  are  present  you  should  inject 
antitoxin  forthwith ;  if  no  suspicious  bacilli  are  seen  it  is  safe  to 
wait  for  the  result  of  the  cultural  examination. 


Interpretation  of  Results. 

The  discovery  of  the  diphtheria  bacillus  in  the  exudate  may  mean — 

{a)  That  the  patient  is  suff'ering  from  diphtheria. 

{b)  That  he  has  sufi"ered  from  diphtheria  and  is  now  con- 
valescent, but  is  still  infectious.  The  bacilli  may  persist  for  weeks 
or  months,  and  while  they  do  so  the  patient  must  be  isolated  and 
treated  with  antiseptic  gargles. 

{c)  That  he  is  in  danger  of  acquiring  diphtheria  if  subjected 
to  any  influence  which  lowers  his  vitality,  or  which  would  cause 
ordinary  sore  throat  in  any  ordinary  person. 

{d)  It  always  means  that  the  person  may  communicate  diphtheria 
to  a  susceptible  subject. 

The  significance  of  Hofi"mann's  bacillus  is  not  yet  settled.  It  is 
frequently  found  in  the  throat  when  the  patient  is  convalescing 
from  an  attack  of  diphtheria,  and  sometimes  in  subjects  who  after- 
wards develop  diphtheria.  It  also  appears  to  cause  epidemics  of 
sore  throat  which  do  not  present  anything  remarkable  in  their 


TETANUS  45 

clinical  characters.  It  is  safest  to  regard  any  patient  who  presents 
these  bacilli  as  being  infectious.  In  other  words,  in  the  present 
state  of  our  knowledge  it  is  best  not  to  draw  any  distinction 
between  Hoffmann's  bacillus  and  the  true  diphtheria  bacillus,  as 
far  as  infectivity  is  concerned." 

A  negative  result  may  mean — 

{a)  That  the  patient  is  not  suffering  from  diphtheria. 

Q))  That  the  swab  did  not  touch  the  affected  area. 

We  exclude  errors  in  technique  and  observation. 

A  sterile  culture  may  mean — 

(a)  That  an  antiseptic  was  used  too  soon  before  taking  the  swab. 

{h)  That  the  diseased  portion  of  the  throat  was  not  touched. 
Other  parts  of  the  mouth  contain  numerous  bacteria,  but  many  of 
them  do  not  grow  well  on  blood-serum  or  ascitic  agar. 

We  again  exclude  errors  arising  in  the  laboratory. 

Whenever  the  culture-tube  remains  sterile,  the  examination 
should  be  repeated. 

TETANUS 

The  pathology  of  tetanus  is  very  much  like  that  of  diphtheria. 
In  each  disease  the  specific  bacilli  are  localized  at  or  near  the 
region  at  which  they  enter  the  body,  and  form  a  toxin  which 
affects  distant  organs.  In  the  case  of  diphtheria,  as  we  have  seen, 
the  toxin  passes  by  the  blood-stream,  but  in  tetanus  it  creeps  from 
the  region  where  it  is  produced  up  the  peripheral  nerves  to  the 
brain  and  spinal  cord.  In  each  case  research  has  shown  that  an 
antitoxin  is  formed  which  neutralizes  this  toxin  and  prevents  it 
from  uniting  with  the  cells  of  the  body,  but  which  has  not  the 
power  of  turning  it  out  from  such  a  combination.  In  other  words, 
tetanus  antitoxin,  like  that  of  diphtheria,  is  preventive,  but  not 
curative.  But  here,  unfortunately,  the  resemblance  between  the 
two  diseases  ceases.  The  local  lesion  in  diphtheria  is  obvious, 
and  its  presence  causes  a  good  deal  of  inconvenience  to  the  patient; 

*  Since  the  publication  of  the  first  edition  of  this  book  there  has  been  much 
controversy  on  this  point,  but  I  have  seen  nothing  which  leads  me  to  alter  the 
above  opinion,  though  it  is  certainly  true  that  laboratory  evidence  of  the 
relation  of  the  organisms  appears  lacking.  It  may  be  noted  that  those  who 
deny  the  relation  of  Hoffmann's  bacillus  to  diphtheria  do  so,  for  the  most  part, 
on  this  failure  of  laboratory  evidence,  whilst  those  who  look  on  Hoffmann's 
bacillus  with  suspicion  do  so  mainly  on  clinical  grounds.  (For  an  account  of 
other  forms  of  sore  throat,  see  p.  105.) 


46  CLINICAL    BACTERIOLOGY    AND    HEMATOLOGY 

he  sees  a  medical  man  early,  and  the  diagnosis  of  diphtheria  is 
made  before  much  of  the  toxin  has  entered  the  blood.  It  is 
different  with  tetanus.  In  this  the  local  symptoms  are  practically 
nil ;  there  may  be  suppuration  at  the  region  of  inoculation,  but 
this  is  so  common  as  not  to  excite  suspicion.  The  result  is  that 
the  diagnosis  is  not  made  until  the  appearance  of  the  symptoms 
referable  to  the  nervous  system  indicates  that  the  period  at  which 
antitoxin  might  have  been  used  with  success  has  gone  by. 

To  illustrate  this  we  will  imagine  the  local  lesion  of  diph- 
theria to  be  so  slight  as  to  be  unnoticed  by  doctor  and  patient. 
The  result  would  be  that  the  disease  would  only  be  diagnosed  when 
the  severe  toxsemic  symptoms  had  manifested  themselves,  and 
antitoxin  would  then  be  almost  or  quite  useless.  If  it  were  not 
for  the  discomfort  and  pain  caused  by  the  throat  lesion  of 
diphtheria,  the  antitoxin  treatment  of  the  disease  would  have 
probably  been  abandoned  as  useless. 

But  tetanus  may  be  diagnosed  by  means  of  a  bacteriological 
examination  of  the  local  lesion  before  toxic  symptoms  have 
appeared,  and  in  cases  where  this  is  done  we  may  safely  look  for 
results  from  tetanus  antitoxin  which  are  as  good  as  those  obtained 
from  the  early  use  of  diphtheria  antitoxin ;  for  the  experimental 
evidence  in  favour  of  the  one  is  every  whit  as  great  as  that  in 
favour  of  the  other. 

Considerations  of  time  would  prohibit  the  bacteriological 
examination  of  the  multitude  of  small  wounds  which  are  seen  by 
the  majority  of  medical  men.  But  a  wound  which  clinical 
experience  and  bacteriological  research  as  to  the  occurrence  of 
tetanus  bacilli  outside  the  body  indicates  as  being  one  which  is 
likely  to  become  infected  with  the  bacillus  in  question  should  be 
submitted  to  a  careful  and  prolonged  search  for  the  bacillus. 
These  are  deep  incised  and  lacerated  wounds,  especially  those  of 
the  hand  and  foot,  and  particularly  if  garden  earth  or  horse-dung 
has  been  rubbed  into  the  tissues.  Wounds  made  with  splinters, 
rusty  nails,  or  the  wads  from  firearms,  should  be  examined. 
Severe  lacerated  and  contused  wounds  in  *' run- over"  cases,  con- 
taminated by  the  dirt  of  the  road,  must  also  be  regarded  with 
suspicion,  for  tetanus  follows  superficial  wounds  almost  as 
frequently  as  deep  ones.  Tetanus  may  follow  a  wound  which 
heals  up  by  first  intention,  but  this  is  unlikely ;  suppuration  or 
necrosis  of  the  edges  (though  not  due  to  the  tetanus  bacillus  itself) 
is  present  in  the  majority  of  cases. 


TETANUS  47 

Examination  of  Pus  from  Suspected  Cases  of  Tetanus. 

Requisites. — i.  Slides  and  cover-glasses. 

2.  A  stiff  platinum  loop. 

3.  Bunsen  burner  or  spirit-lamp.  _ 

4.  Loffler's  blue  or  carbol  thionin. 

5.  Materials  for  Gram's  staining. 

6.  Balsam. 

If  cultures  are  to  be  taken,  add  a  pipette  (see  Fig.  146),  a  deep 
tube  of  agar  to  which  2  per  cent,  of  grape-sugar  has  been  added 
previous  to  sterilization,  a  flask  of  water,  and  a  thermometer. 

Method, — Scrape  the  deeper  portions  of  the  wound  with  the 
platinum  loop,  and  spread  out  the  secretion  thus  obtained  on  the 
surface  of  a  slide.  Prepare  several  of  these  slides,  and  fix  the 
film  by  heat.  Stain  some  by  the  simple  stain  for  two  minutes 
and  others  by  Gram's  method. 

The  bacillus  of  tetanus  is  about  as  long  as  the  tubercle  bacillus, 
and  is  very  slender.  It  stains  by  Gram's  method.  A  very 
characteristic  feature  is  its  method  of  spore- formation.  The 
spores  are  spherical  bodies  which  are  formed  at  the  extremities  of 
the  bacilli,  giving  them  the  appearance  of  pins  or  drumsticks. 
The  spores  do  not  stain  by  the  ordinary  stains,  and  appear  as 
colourless  and  highly  refractile  bodies  (Plate  II.,  Fig.  i). 

The  cultures  are  made  in  agar  to  which  2  per  cent,  of  grape- 
sugar  is  added,  and  the  needle  or  pipette  used  in  making  the 
inoculation  is  plunged  deep  down  into  the  medium.  The  bacillus 
of  tetanus  is  an  anaerobe — i.e.,  it  grows  only  in  the  absence  of 
oxygen.  The  stabs  are  made  deep  in  order  to  inoculate  the 
material  far  away  from  the  air,  and  the  glucose  is  added  to  absorb 
any  oxygen  which  may  be  in  the  medium.  To  increase  our 
chances  of  obtaining  this  bacillus  in  pure  culture,  the  material 
to  be  examined  is  to  be  heated  to  a  temperature  which  will  kill  all 
developed  bacteria,  but  which  will  not  be  injurious  to  spores ;  the 
tetanus  bacillus  is  the  only  anaerobic  organism  with  a  spherical 
terminal  spore  which  is  at  all  likely  to  occur  in  a  wound. 

Method. — The  inoculations  are  to  be  made  with  a  pipette.  If 
the  pus  which  comes  from  the  wound  can  be  drawn  up  into  the 
capillary  tube  of  a  glass  pipette  such  as  is  described  on  p.  33, 
the  material  should  be  collected  in  this  way.  If  this  is  not  the 
case,  the  wound  must  be  scraped  with  a  sterilized  platinum 
needle  or  other  suitable  instrument,  and  the  material  thus  obtained 


48  CLINICAL    BACTERIOLOGY    AND    H.F.MATOLOGY 

mixed  with  some  boiled  water  (previously  cooled)  and  then 
sucked  up  into  the  pipette ;  the  end  of  the  latter  is  then  to  be 
sealed  in  the  flame,  care  being  taken  that  the  material  itself  is 
not  heated. 

Having  filled  and  sealed  the  pipette,  heat  some  water  in  a  small 
flask  or  large  test-tube  until  it  reaches  80°  C,  as  measured  by  the 
thermometer ;  insert  the  sealed  end  of  the  pipette  in  the  water, 
and  maintain  the  temperature  for  ten  minutes.  The  thermometer 
is  to  be  kept  in  the  water  the  whole  of  the  time,  and  the  flame  is 
to  be  taken  away  when  the  temperature  rises  above  80°  C,  and 
reapplied  when  it  falls  below  that  point. 

At  the  end  of  this  time  the  pipette  will  contain  no  living  object 
other  than  spores.  Break  off  its  point  and  insert  it  gently  into 
the  glucose  agar,  taking  care  to  keep  exactly  in  the  axis  of  the 
tube,  until  the  tip  of  the  pipette  reaches  almost  to  the  bottom  of 
the  test-tube.  Withdraw  the  pipette  gradually,  blowing  out  its 
contents  as  you  do  so.  The  spores  of  the  tetanus  bacillus  (if 
present)  will  now  be  inoculated  deep  down  in  the  medium,  far 
away  from  the  air.  To  reduce  the  supply  of  oxygen  still  further 
it  is  a  good  plan  to  melt  some  paraffin  (a  hard  candle  answers 
perfectly)  and  pour  a  layer  an  inch  thick  over  the  surface  of  the 
medium ;  or  vaseline  which  has  been  heated  to  the  boiling-point 
of  water  (to  sterilize  it)  may  be  used. 

The  cultures  thus  made  are  to  be  incubated  for  a  few  days  at 
the  body  temperature.  After  about  forty-eight  hours  the  growth 
begins  to  appear  in  the  deeper  portions  of  the  tube  as  a  series  of 
delicate  wavy  outgrowths  from  the  central  stab.  These  do  not 
appear  in  the  upper  portion  of  the  medium,  where  the  oxygen 
(which  diff"uses  down  from  the  air,  unless  the  tube  has  been 
sealed  with  paraffin)  hinders  their  growth.  If  the  culture  is  pure 
— and  this  is  not  likely  to  be  the  case,  as  these  wounds  are 
usually  very  highly  contaminated — no  gas  will  be  produced  by  the 
growth,  whereas  the  bulk  of  the  other  organisms  which  are 
likely  to  occur  produce  gas.  A  bubble  or  two  in  the  course 
of  the  growth,  therefore,  indicates  that  the  culture  is  not  pure 
tetanus  bacillus,  but  that  organism  may  nevertheless  be  present. 
If  the  tube  shows  such  a  growth,  it  should  be  submitted  to  a 
microscopic  examination.  It  is  a  good  plan  to  break  the  tube 
and  to  spHt  up  the  cylinder  of  medium  with  a  knife ;  films  are 
made  from  the  growth  and  stained  as  above.  Spores  are  formed 
after  about  thirty-six  hours. 


THE    PNEUMOCOCCUS,    PNEUMONIA,    ETC.  49 

The  other  methods  of  cultural  examination  are  far  more 
difficult. 

Interpretation  of  Results. 

If  bacilli  having  the  above  characters  are  found  in  films,  the 
diagnosis  of  tetanus  must  not  be  considered  as  being  absolutely 
proven,  for  there  are  other  bacilli  which  might  be  mistaken  for 
those  under  discussion ;  but  the  probability  that  the  patient  will 
develop  the  disease  is  so  strong  that  steps  should  be  taken 
accordingly.  The  wound  should  be  scraped  and  thoroughly 
treated  with  antiseptics,  and  antitoxin  should  be  given.  If  the 
deeper  portion  of  the  glucose-agar  stab  shows  the  tree-like 
growth  which  has  been  described  and  contains  slender  drum- 
stick bacilli,  the  case  is  strengthened,  even  although  the  upper 
part  of  the  medium  is  contaminated  with  other  organisms. 

The  only  way  in  which  the  bacilli  can  be  recognized  with 
absolute  certainty  is  by  means  of  animal  experiments ;  this  is  a 
very  certain  method,  and  one  that  can  be  carried  out  in  the 
absence  of  pure  cultures.  The  material  is  diluted  with  some 
broth  and  divided  into  two  parts,  of  which  one  is  injected  into  an 
animal  just  as  it  is,  whilst  the  other  is  mixed  with  antitetanic 
serum  and  then  injected.  In  a  case  of  tetanus,  the  first  animal 
will  die  with  tetanic  symptoms,  whilst  the  latter  will  survive  or 
die  of  sepsis  without  tetanus. 


THE  PNEUMOCOCCUS,  PNEUMONIA,  ETC. 

The  pneumococcus  is  a  very  important  organism,  and  one 
which  plays  a  prominent  part  in  the  production  of  disease.  It 
may  occur  in  the  mouth  in  a  healthy  person ;  hence  its  recogni- 
tion in  small  quantities  in  the  sputum  is  not  of  diagnostic  value. 

The  pneumococcus  is  a  very  common  cause  of  disease  of  the 
respiratory  system.     It  causes  : 

1.  Acute  lohav pneumonia.  Opinions  differ  as  to  whether  it  is  the 
only  cause  of  this  disease,  though  it  appears  most  probable  that 
this  is  the  case. 

2.  Lohulav  {broncho-)  pneumonia.  This  disease  may  also  be 
caused  by  streptococci,  staphylococci,  diphtheria  bacilli,  influenza 
bacilli,  plague  bacilH,  tubercle  bacilli,  and  others.  The  pneumo- 
coccus may  also  occur  as  a  secondary  infection  in  lobular  pneumonia 
due  to  any  of  these. 

4 


50  CLINICAL    BACTERIOLOGY   AND    HiEMATOLOGY 

3.  Pleurisy,  either  the  serous,  fibrinous,  or  purulent  varieties, 
and  especially  in  children.  It  is  important  to  notice  that  the 
prognosis  of  empyema  is  better  when  the  disease  is  due  solely 
to  the  pneumococcus  than  when  other  organisms  (streptococci, 
staphylococci,  tubercle  bacilli,  etc.)  are  present,  and  such  cases 
usually  recover  without  resection  of  ribs.  The  bacteriological 
examination  of  the  pus  from  a  pleural  cavity  may  thus  lead  to 
results  important  as  to  prognosis  and  treatment. 

4.  The  pneumococcus  may  occur  as  a  secondary  infection  in 
almost  any  disease  of  the  lung  ;  for  instance,  in  the  walls  of  a 
phthisical  vomica. 

The  most  important  primary  lesions  due  to  the  pneumococcus 
outside  the  respiratory  system  are  : 

1.  Otitis  media  (of  which  it  is  a  very  common  cause),  empyema 
of  the  antrum,  and  of  the  frontal  and  other  accessory  sinuses  of 
the  nose. 

2.  Erysipelas,  which  is  usually  due  to  another  organism,  the 
Streptococcus  pyogenes. 

3.  Brawny  induration  of  the  skin,  with  or  without  suppuration. 
This  is  uncommon,  but  I  have  seen  several  cases. 

4.  Suppuration  in  any  region,  especially  in  infants,  in  whom  it 
is  probably  the  most  common  pyogenic  organism. 

Pneumococci  may  escape  into  the  blood  from  any  of  these 
lesions,  and  may  appear  in  that  fluid  when  there  is  no  obvious 
primary  lesion  from  which  it  could  have  gained  access.  The 
most  common  results  are  : 

1.  Septicemia. 

2.  Ulcerative  endocarditis.  Many  other  bacteria  may  cause  this 
disease. 

3.  Meningitis.  Pneumococcal  meningitis  may  also  be  due  to 
direct  spread  from  the  middle  ear. 

4.  Arthritis. 

5.  Peritonitis. 

The  prognosis  in  these  latter  affections  is  on  the  whole  slightly 
better  if  they  are  due  to  the  pneumococcus  than  if  caused  by 
other  organisms,  except  in  the  case  of  meningitis,  which  is  usually 
fatal  in  a  few  days. 

In  actual  practice  we  have  most  commonly  to  search  for  the 
pneumococcus  in  sputum,  pus,  and  blood.  In  the  latter  case 
cultural  methods  are  usually  necessary,  and  we  shall  defer  its 
consideration  for  the  present. 


I 


THE    PNEUMOCOCCUS,    PNEUMONIA,    ETC.  5I 

Sputum. — The  examination  of  sputum  may  be  made  in  order  to 
make  a  diagnosis  as  to  the  presence  or  absence  of  pneumonia,  in 
a  case  in  which  the  physical  .signs  are  indeterminate,  or  to 
estabHsh  the  nature  of  a  lobular  pneumonia. 

The  patient  must  wash  out  his  mouth  with  water,  which  should 
have  been  boiled  and  allowed  to  cool.  He  must  then  spit  into  a 
clean  wide-mouthed  bottle,  also  containing  boiled  water,  and  care 
must  be  taken  that  the  sputum  used  for  the  examination  comes 
directly  from  the  lungs,  and  is  not  merely  mucus  which  has 
collected  in  the  mouth. 

The  mass  of  mucus  forming  a  single  "  spit "  is  agitated  gently 
in  the  water  to  remove  contaminations  from  the  bronchial  tubes 
and  mouth ;  the  water  is  poured  off  and  more  added,  and  the 
process  repeated  several  times.  Then  the  mass  of  mucus  is  fished 
out,  placed  in  a  watch-glass,  carefully  opened  with  a  pair  of  scissors, 
and  a  piece  about  as  big  as  a  pea  is  removed  from  the  centre  of 
the  mass  with  a  platinum  loop.  It  is  placed  on  a  clean  slide, 
another  slide  pressed  upon  it,  and  the  two  are  slid  apart.  The 
films  thus  obtained  are  allowed  to  dry,  and  fixed  by  heat  in  the 
usual  way. 

One  is  then  stained  by  Gram's  method,  and  counterstained  by 
dilute  carbol  fuchsin  for  about  a  quarter  of  a  minute,  washed, 
dried,  and  mounted,  and  the  other  with  undiluted  carbol  fuchsin. 

The  pneumococcus  is  a  diplococcus — i.e.,  the  individual  cocci 
are  arranged  in  pairs.  Each  coccus  has  usually  an  oval  or  lancet 
shape,  the  sharp  ends  of  the  two  germs  pointing  away  from  one 
another  (Plate  I.,  Fig.  3,  and  Plate  III.,  Fig.  4).  Abnormal 
forms  (round  cocci,  short  bacilli,  etc.)  are  frequent.  The  pneumo- 
coccus has  a  capsule  when  it  occurs  in  the  living  body  or  in  patho- 
logical exudates,  but  not  in  most  cultures.  This  capsule  does  not 
stain  readily,  and  appears  in  a  properly-stained  specimen  as  a 
clear  halo  round  the  two  cocci. 

Examine  your  Gram  specimen  first.  The  pneumococci  (which 
will  be  almost  black)  should  be  clearly  seen,  and  you  should  be  able 
to  make  out  their  shape  and  characteristic  arrangement  in  pairs. 

The  general  surface  of  the  film  is  stained  pink,  while  there  is  a 
clear  and  colourless  zone  round  each  pair  of  cocci.  This  is  the 
capsule,  which  is  rendered  distinct  by  "negative  staining,"  and 
these  appearances  will  be  better  seen  in  a  somewhat  thick  and 
deeply-stained  film,  though  if  the  carbol  fuchsin  has  been  allowed 
to  act  too  long  the  capsule  may  be  stained  a  faint  pink.     There 

4—2 


52  CLINICAL    BACTERIOLOGY   AND    H^EMATOLOGY 

are  special  methods  of  staining  which  may  be  used  to  render  the 
capsule  more  distinct,  but  these  are  usually  unnecessary  for  the 
diagnosis. 

Interpretation  of  Results. 

In  cases  of  lobar  pneumonia  you  will  probably  fmd  pneumococci 
in  great  quantity,  and  no  other  bacteria  in  a  specimen  of  sputum 
made  in  the  manner  descvihed.  If  you  find  many  pneumococci  in  a 
case  of  lobular  pneumonia  the  disease  may  have  been  caused  by 
another  germ,  and  the  cocci  in  question  may  have  been  nothing 
more  than  a  secondary  infection.  A  specimen  should  be  stained 
deeply  by  carbol  fuchsin  or  methylene  blue  and  searched  for 
bacilli  resembling  those  of  influenza,  etc.,  and  another  should  be 
stained  for  the  tubercle  bacillus  if  the  clinical  aspect  of  the  case 
suggests  the  possibility  of  a  tuberculous  origin  for  the  disease. 

Pus  is  examined  in  the  same  way,  and  presents  similar  appear- 
ances. Most  of  the  cocci  are  extracellular,  but  some  are  frequently 
contained  in  the  cells,  and  may  then  not  retain  Gram's  stain. 

It  is  not  usually  necessary  to  make  cultures,  as  the  pneumococcus 
is  readily  recognized  in  pathological  material  from  its  morphological 
appearance  and  staining  reactions  alone.  Where  cultures  are 
required,  agar  is  about  as  good  a  medium  as  can  be  used,  and  it 
must  be  incubated  at  the  body  temperature.  The  colonies  are 
visible  after  twenty-four  hours  as  very  small  transparent  circular 
masses,  which  are  but  slightly  raised  and  show  but  little  tendency 
to  increase  in  size  on  further  incubation.  It  is  a  very  delicate 
organism,  and  one  which  readily  dies  out  on  ordinary  media,  so 
that  if  a  culture  is  to  be  "  kept  going  "  it  must  be  transplanted 
every  two  or  three  days  to  a  fresh  tube.  This  is  one  of  its  most 
characteristic  features. 

INFLUENZA 

In  the  first  edition  of  this  book,  in  treating  of  influenza,  I  followed 
the  usual  teaching  of  bacteriologists  and  regarded  the  disease  as  a 
specific  one,  and  as  being  caused  in  all  cases  by  the  influenza  (or 
Pfeiff"er's)  bacillus.  Recent  observations,  both  in  England  and  on 
the  Continent,  have  shown  that  this  view  can  no  longer  be  upheld, 
unless  we  greatly  restrict  the  use  of  the  term  "  influenza,"  and  use 
it  only  for  those  cases  in  which  the  bacillus  in  question  is  found. 
This  is  quite  unjustifiable,  for  the  diseases  appear  to  be  identical 
in  clinical  history  ;  and  in  cases  in  which  we  should  be  practically 


INFLUENZA  ^  53 

certain  of  finding  Pfeiffer's  bacillus  if  they  occurred  a  few  years 
back,  we  now  find  other  organisms,  especially  the  Micrococcus 
catarrhalis.  In  this  uncertain  state  of  bacteriology  the  results  of 
an  examination  of  the  sputum  are  deprived  of  much  of  their  value 
as  a  means  of  diagnosis,  but  the  methods  will  be  described,  since 
more  information  is  required,-  and  this  information  the  general 
practitioner  is  usually  in  the  best  situation  to  obtain. 

Methods. — If  possible,  the  sputum  should  be  obtained  in  a  method 
similar  to  that  recommended  in  pneumonia,  as  it  greatly  facilitates 
the  process  and  renders  the  results  more  trustworthy  if  the  sputum 
comes  directly  from  the  lungs,  and  is  not  contaminated  with  bacteria 
from  the  mouth.  The  nasal  secretion  may  also  be  examined,  and 
frequently  contains  the  organisms  in  pure  culture  and  vast  numbers; 
where  the  nose  is  affected,  better  results  will  be  obtained  in  this 
way  than  from  the  sputum.  The  mucus  may  be  collected  on  a 
diphtheria  swab  or  on  a  platinum  loop,  or  by  means  of  one  of  the 
angled  pipettes  described  on  p.  no,  though  it  is  often  too  thick  to 
be  sucked  up  into  such  a  narrow  tube. 

Films  are  prepared  from  the  sputum  by  squeezing  a  small  mass 
between  two  slides  and  sliding  them  apart.  Dry  and  fix  by  heat. 
Two  should  be  prepared.  The  first  should  be  stained  by  Gram's 
method,  and  counterstained  by  dilute  carbol  fuchsin  for  a  quarter 
of  a  minute,  then  washed,  dried,  and  mounted.  The  other  is  to  be 
stained  more  deeply  with  carbol  fuchsin  or  Loffler's  blue — about 
five  minutes  with  gentle  heat  in  either  case.  The  influenza  bacillus 
stains  with  difficulty,  and  may  not  be  seen  in  the  Gram's  specimen 
which  is  lightly  stained  with  fuchsin.  And  there  is  a  good  reason 
for  not  carrying  the  counterstaining  too  far  in  the  former  case, 
since  if  the  carbol  fuchsin  is  used  for  too  long  a  time  it  may  dis- 
place the  violet  stain  of  an  organism  which  retains  Gram. 

The  influenza  bacillus  is  an  extremely  minute  organism,  and  one 
which  requires  the  highest  powers  of  the  microscope  for  its  study. 
It  is  an  extremely  minute  rod,  and  it  would  take  from  eight  to 
sixteen  of  these  rods  to  make  up  the  diameter  of  a  red  blood- 
corpuscle.  They  occur  in  vast  numbers  in  the  sputum  or  nasal 
mucus,  and  are  frequently  found  within  the  leucocytes,  and  when 
in  this  situation  may  appear  to  have  a  capsule,  being  contained  in 
vacuoles  in  the  protoplasm  (Plate  II.,  Fig.  3).  They  are  often 
arranged  in  pairs,  in  which  case  they  might  be  mistaken  for  small 
but  unusually  elongated  pneumococci,  but  for  the  fact  that  they  do 
not  stain  by  Gram's  method. 


54  CLINICAL    BACTERIOLOGY    AND    HEMATOLOGY 

These  features  are  sufficient  to  identify  them  for  dinical 
purposes.  Cultures  are  difficult  to  obtain,  since  the  organism 
only  grows  in  presence  of  haemoglobin — e.g.,  on  agar  tubes  streaked 
with  sterile  blood  (see  p.  85).  Under  these  circumstances  they 
form  very  minute  translucent  colonies,  much  like  those  of  the 
pneumococci,  and  cultures  have  a  great  tendency  to  die  out. 

The  M.  catarrhalis  (Plate  III.,  Fig.  5),  the  next  most  frequent 
cause  of  clinical  influenza,  is  a  diplococcus  which  does  not  stain  by 
Gram,  and  which  has  a  considerable  amount  of  resemblance  to 
the  other  two  non-Gram-staining  diplococci,  the  gonococcus  and  the 
meningococcus.  It  occurs  in  vast  numbers  in  the  sputum  and 
nasal  mucus  of  influenza,  in  the  nasal  mucus  of  a  "  common  cold," 
and  is  a  common  cause  of  bronchitis  of  ordinary  type.  It  is  also 
an  occasional  cause  of  sore  throat,  and  is  not  infrequently  met 
with  in  the  examination  of  supposed  cases  of  diphtheria.  The 
resemblance  to  the  two  other  organisms  named  arises  from  the 
fact  that  it  is  frequently  intracellular.  There  is  not  usually  any 
difficulty  in  distinguishing  between  the  three,  owing  to  the  difference 
in  their  habitat — the  gonococcus  affecting  the  mucous  membrane 
of  the  urethra  or  cervix,  the  meningococcus  the  meninges,  and 
the  M.  catarrhalis  the  nose,  mouth,  and  respiratory  passages. 
There  are  minute  morphological  differences  between  the  three, 
and  an  expert  can  usually  identify  them  in  film  preparations 
from  the  body,  but  where  there  is  any  question  of  the  nature  of  the 
organism  present  cultures  ought  to  be  made.  The  M.  catarrhalis 
is  the  only  one  of  the  three  which  will  grow  on  gelatin  at  the 
room  temperature. 

ANTHRAX 

Anthrax  occurs  in  man  in  three  forms.  The  most  common  is 
cutaneous  anthrax,  or,  as  it  is  sometimes  called,  malignant  pustule. 
Pulmonary  anthrax,  or  wool- sorter's  disease,  is  much  rarer,  and 
intestinal  anthrax  rarer  still.  The  practitioner  will  find  the  greatest 
assistance  from  a  bacteriological  examination  in  the  cutaneous 
form  of  the  disease ;  he  may  search  for  the  specific  bacillus  in 
the  sputum  in  a  supposed  case  of  wool-sorter's  disease,  but  he 
must  be  careful  in  his  interpretation  of  his  result,  as  bacilli  which 
might  be  mistaken  by  an  untrained  observer,  relying  on  the 
morphological  appearances  alone,  sometimes  occur  in  the  sputum. 
The  search  for  bacilli  in  the  faeces  in  a  supposed  case  of  intestinal 
anthrax  must  be  relegated  to  an  expert. 


ANTHRAX  55 

The  true  nature  of  a  case  which  is  examined  post-mortem  can 
easily  be  determined  bacteriologically ;  the  cut  surface  of  the 
liver  or  spleen  should  be  rubbed  upon  a  clean  slide,  and  the  films 
treated  secundum  art  em.  They  will  probably  show  the  bacilli  in 
large  numbers.  Sections  may  also  be  cut  and  stained  by  Gram's  - 
method  (see  p.  172). 

In  the  later  stages  of  any  infection  with  anthrax  the  bacilli 
may  be  found  in  the  blood.  They  may  be  apparent  on  examina- 
tion of  stained  films,  or  by  cultural  methods  similar  to  those  used 
in  the  diagnosis  of  malignant  pustule. 

The  anthrax  bacillus  varies  considerably  in  length,  but  is 
always  a  large  organism,  and  may  be  considerably  longer  than 
the  diameter  of  a  red  blood-corpuscle.  It  is  much  thicker  than 
the  bacilli  which  have  been  dealt  with  hitherto,  and  it  is  invariably 
straight.  The  ends  of  these  bacilli  are  cut  sharply  at  right  angles 
to  the  sides  of  the  organism,  and  may  be  even  somewhat  concave  ; 
this  is  a  most  characteristic  feature.  The  anthrax  bacillus  stains 
by  Gram's  method  (Plate  I.,  Fig.  4). 

In  cultures  the  appearances  are  somewhat  different.  Here  the 
bacilli  are  frequently  arranged  in  long  chains  which  have  an 
appearance  which  has  been  compared  to  that  of  a  bamboo  ;  chains 
occur  in  the  blood  or  in  the  inflammatory  exudate,  but  are  usually 
much  shorter  than  those  seen  in  cultures.  But  the  most  important 
features  in  cultures  of  the  anthrax  bacillus  is  the  development 
of  spores,  which  are  oval,  highly  refractile  bodies,  and  lie  in  or 
near  the  centre  of  the  bacilli,  one  in  each.  These  spores  are 
possessed  of  tough  capsules,  which  resist  the  action  of  the  ordinary 
stains  much  in  the  same  way  as  the  tubercle  bacillus.  Thus 
it  happens  that  in  films  of  a  cultivation  of  the  anthrax  bacillus 
which  have  been  stained  with  such  a  dye  as  weak  methylene  blue 
the  spores  are  readily  seen  as  colourless  and  refractile  oval  areas 
in  the  centre  of  the  bacilli,  the  latter  being  stained  blue.  The 
spores  themselves  may  be  stained  by  a  modification  of  the 
process  used  for  the  tubercle  bacillus.  The  films  are  first  stained 
by  heated  carbol  fuchsin,  which  penetrates  slowly  through  the 
capsule;  they  are  then  decolorized  by  a  very  rapid  immersion 
in  very  dilute  sulphuric  acid  (i  per  cent.)  or  in  methylated  spirit, 
and  examined  microscopically.  If  the  red  colour  has  been  entirely 
removed  from  the  bacilli,  but  is  still  present  in  the  spores,  the  films 
are  ready  to  be  counterstained  by  methylene  blue  ;  if  not,  they 
must  be  dipped  in  the  acid  or  spirit  once  more  and  re-examined, 


56  CLINICAL    BACTERIOLOGY   AND    HiEMATOLOGY 

When  this  process  is  successful,  the  spores  are  stained  red,  and 
the  bacilli  blue  (Plate  I.,  Fig.  4). 

The  presence  of  spores  enables  us  to  isolate  the  bacilli  from  most 
of  the  organisms  with  which  they  are  likely  to  be  contaminated, 
by  a  very  simple  process.  The  spores  resist  the  action  of  heat 
just  as  they  resist  stains,  and  for  the  same  reason,  and  a  suitable 
temperature  will  kill  off  all  the  non-sporing  organisms  and  spare 
the  spores.  The  latter  may  then  be  inoculated  at  a  suitable 
temperature,  and  will  develop  into  bacilli.  This  process,  however, 
is  not  applicable  to  the  examination  of  the  blood  or  morbid 
effusions,  as  the  bacillus  of  anthrax  does  not  form  spores  in  the 
living  body.  In  this  it  differs  from  the  tetanus  bacillus,  in  which 
the  process  may  be  applied  direct  to  the  material  from  the  body. 

Investigation  of  a  Supposed  Case  of  Malignant  Pustule. 

Requisites. — i.  Several  glass  pipettes;  if  cultures  are  not 
required,  one  will  be  enough. 

2.  Clean  slides  and  cover-glasses. 

3.  Bunsen  burner  or  spirit-lamp. 

4.  Loffler's  methylene  blue ;  also  the  materials  for  Gram's 
staining. 

5.  Balsam. 

6.  Tubes  of  gelatin  if  cultivations  have  to  be  taken. 

Method, — Break  off  the  extreme  tip  of  one  of  the  glass  pipettes 
and  insert  into  one  of  the  vesicles  around  the  dark  papule  in  the 
centre  of  the  lesion  ;  it  may  be  necessary  to  make  a  puncture 
with  a  sterilized  needle  before  this  can  be  done.  If  the  fluid  does 
not  rise  spontaneously  into  the  pipette,  break  off  the  other  end 
and  suck  gently,  watching  the  column  of  fluid  so  that  it  does  not 
get  into  your  mouth.  It  is  safer  to  use  a  pipette  with  a  wide 
mouthpiece  plunged  with  cotton-wool  (Fig.  14),  or,  best  of  all, 
to  suck  up  the  fluid  by  means  of  an  india-rubber  nipple. 

Having  obtained  a  drop  or  two  of  the  fluid  exudate,  blow  it  out 
on  to  the  surface  of  a  clean  slide  and  spread  it  out  into  a  film ; 
prepare  as  many  of  these  as  you  can.  Allow  them  to  dry,  and 
stain  one  with  Loffler's  blue  and  some  by  Gram's  method. 

Examine  with  the  oil-immersion  lens.  Make  a  careful  search 
over  the  films,  looking  for  large  cigarette-shaped  bacilli,  noting 
whether  they  are  or  are  not  arranged  in  chains.  Examine  the 
Gram  specimens,  and  see  whether  the  bacilli  are  to  be  seen  in 
them  also. 


ANTHRAX  57 


Interpretation  of  Results. 

If  the  case  is  really  one  of  malignant  pustule,  the  chances  are 
very  greatly  in  favour  of  your  finding  the  bacilli  in  large  numbers, 
and  the  failure  to  do  so  tells  strongly  against  a  positive  diagnosis. 

Cultural  Methods. — The  fluid  for  examination  is  taken  in  exactly 
the  same  way  as  that  described  above,  but  the  isolation  of  the 
organisms  will  be  greatly  facilitated  if  antiseptic  methods  are 
employed  to  prevent  contamination  with  skin  bacteria.  To  this 
end  the  surface  of  the  lesion  should  be  washed  gently  with 
carbolic  or  perchloride  lotion,  and  then  (very  thoroughly)  with 
alcohol  or  methylated  spirit  to  remove  the  antiseptic.  The 
surface  is  then  allowed  to  dry. 

If  the  material  is  to  be  transmitted  to  a  pathologist  for  examina- 


FiG.  19. — Petri's  Dish. 

tion  (and  this  is  the  wisest  course  to  adopt,  as  animal  inoculations 
are  almost  necessary  for  the  absolute  identification  of  the  bacillus), 
the  fluid  must  be  carefully  sucked  up  into  the  bulb,  and  both  ends 
of  the  pipette  carefully  sealed. 

If  the  examination  is  to  be  made  at  home,  the  best  way  is  to 
make  two  inoculations  in  gelatin.  The  first  should  be  a  stab 
culture,  and  may  be  made  with  the  pipette  direct ;  or  the  fluid 
may  be  blown  out  into  a  watch-glass  or  on  to  the  surface  of  a 
slide  (in  either  case  sterilized  by  being  heated  in  the  flame  and 
then  allowed  to  cool),  and  the  stab  made  by  dipping  the  end  of  a 
straight  platinum  needle  into  the  fluid,  and  then  driving  it  into  the 
gelatin. 

The  other  culture  is  made  with  the  pipette ;  this  is  driven  into 
the  gelatin  in  a  tube  and  the  contents  blown  out.  The  gelatin  is 
then  immersed  in  warm  water  until  it  is  melted,  and  poured  out 
into  a  Petri's  dish  (Fig.  19)  previously  sterilized  by  dry  heat  and 
allowed  to  cool. 

Both  cultures  are  incubated  at  a  temperature  of  about  20°  C. 

In  about  two  days   the   gdatin   stab-tube  will   show  a  very 


58 


CLINICAL    BACTERIOLOGY    AND    HEMATOLOGY 


characteristic  appearance  if  the  anthrax  bacilli  are  present  in 
pure  culture.  The  growth  takes  place  in  Hnes  which  project 
nearly  at  right  angles  to  the  line  of  inoculation,  and  grow  more 
vigorously  the  nearer  they  are  to  the  surface.  The  result  is  the 
development  of  a  culture  which  has  a  strong  resemblance  to  an 
inverted  fir-tree  (Fig.  20).  In  another  day  or  two  the  gelatin  will 
begin  to  show  a  certain  amount  of  liquefaction,  which  begins  at 
the  surface. 

The  appearances  in  the  plate  culture  are 
perhaps  not  quite  so  characteristic,  but  they 
are  manifested  in  impure  cuhures.  The 
young  colonies  of  anthrax  bacilli  have  a 
whorled   appearance,  which  has   been   com- 


FlG.  21. 


-Young  Colony  of  Anthrax  Bacillus 
(x  15).     (Crookshank.) 


Fig.  20. -Stab  Cul-   p^red  to  a  barrister's  wig  or  to  the  head  of 

TURE    OF  Anthrax    ^  '=' 

Bacillus.    (Crook-    Medusa  (Fig.  21).    The  plate  should  be  placed 

shank.)  upon    the    stage    of    the    microscope,    and 

examined  for  these  colonies  with  the  low 
power.  If  one  is  found,  a  clean  cover-glass  should  be  pressed 
upon  it,  lifted  up  with  a  needle,  so  as  to  bring  up  the  colony  with 
it,  fixed  by  heat,  and  stained  with  carbol  thionin  or  methylene 
blue.  The  colonies  are  most  characteristic  after  two  days' 
incubation  ;  at  a  later  period  the  gelatin  is  liquefied  and  spores 
are  formed. 

A  culture  which  presents  these  cultural  and  morphological 
appearances  may  be  considered  to  be  one  of  anthrax  with  almost 
absolute  certainty,  though  other  tests  (notably  animal  tests)  would 
be  applied  in  a  laboratory. 


TUBERCLE  59 


TUBERCLE 


The  diagnosis  of  tuberculosis  by  bacteriological  methods  (in 
the  case  of  most  morbid  exudates)  is  within  the  reach  of  every 
practitioner ;  cultural  methods  are  not  used,  and  the  recognitioiT 
of  the  bacillus  is  rapid,  easy,  and  certain. 

The  bacilli  may  be  sought  for  in  sputum,  urine,  pus,  faeces,  or 
any  other  morbid  material.  We  will  first  describe  the  method  of 
staining  which  should  be  adopted,  then  the  characters  on  which 
the  recognition  of  the  bacillus  depends,  and,  lastly,  the  methods 
by  which  the  films  are  prepared  from  the  various  materials. 

Staining  the  Tubercle  Bacillus. 

Requisites. — i.  Slides,  cover-glasses,  forceps,  and  balsam. 

2.  A  Bunsen  burner  or  spirit-lamp. 

3.  Dilute  sulphuric  acid — about  20  per  cent. — contained  in  a 
wide-mouthed  bottle  or  in  a  jar.  This  must  be  large,  enough  to 
admit  a  slide,  but  not  large  enough  to  permit  it  to  fall  down  to 
the  bottom. 

4.  Carbol  fuchsin. 

5.  Methylene  blue.   (Saturated  watery  solution  or  Loffler's  blue.) 

6.  A  metal  (iron  or  copper)  plate.  The  exact  dimensions  do 
not  matter,  but  8  by  4  by  ^  inches  is  a  convenient  size.  It  should 
be  mounted  upon  a  tripod.  This  slab  is  not  absolutely  necessary, 
but  it  is  a  very  great  advantage. 

Method. — We  will  suppose  that  the  film  has  been  prepared  by 
one  of  the  methods  described  subsequently,  allowed  to  dry,  and 
fixed  by  heat. 

1.  Place  the  slide  upon  the  metal  plate,  and  heat  the  latter  by 
the  flame.  Flood  the  slide  with  carbol  fuchsin,  and  let  the  heat 
continue  until  the  stain  steams,  but  do  not  allow  it  to  dry  up ;  let 
this  go  on  for  from  three  to  five  minutes.  If  the  stain  shows 
signs  of  drying  up,  add  a  little  more ;  if  it  begins  to  boil,  slide  it 
along  the  plate  away  from  the  flame,  or  remove  the  latter  for  a 
short  time. 

If  you  have  no  metal  plate,  it  is  possible  to  hold  the  slide  with 
a  pair  of  forceps,  but  in  this  case  the  film  is  most  conveniently 
made  on  a  cover-glass. 

Remember  not  to  let  the  stain  dry  up. 

2.  Remove  the  slide  from  the  plate  with  the  forceps,  and  wash 
it  under  the  tap  or  in  a  bowl  of  water. 


6o  CLINICAL    BACTERIOLOGY   AND    HEMATOLOGY 

3.  Put  it  into  the  bottle  containing  the  dilute  acid.  After  three 
or  four  minutes  withdraw  it  and  again  wash.  If  much  pink 
colour  comes  back,  re-insert  it  in  the  acid  for  a  short  time,  and 
again  wash.  The  process  must  be  repeated  until  the  film  only 
shows  a  slight  pink  tinge. 

4.  Now  apply  the  methylene  blue  for  half  a  minute  or  so. 

5.  Wash,  dry  with  blotting-paper,  and  then  by  gentle  heat. 
Apply  a  drop  of  balsam,  and  cover. 

Another  Method. — Filter  into  a  test-tube  sufficient  carbol  fuchsin 
to  flood  the  film,  boil  it  over  the  Bunsen  or  spirit-lamp,  and  pour 
it  on  to  the  film  whilst  still  boiling.  Let  it  act  until  it  is  cool, 
when  the  bacilli  will  be  found  to  be  stained,  and  the  process  of 
decolorization  may  be  proceeded  with. 

The  times  which  are  given  above  may  be  considerably  shortened 
in  practice,  but  I  do  not  advise  this  until  considerable  skill  is 
acquired.  Bacilli  are  much  more  easy  to  recognize  if  they  are 
deeply  stained  ;  this  is  the  reason  for  the  prolonged  staining, 
which  may  appear  unnecessary  to  some.  The  prolonged  de- 
colorization is  an  advantage,  since  it  insures  that  the  tubercle 
bacilli  shall  be  the  only  things  left  stained  red  ;  if  you  leave  the 
preparation  in  the  acid  for  a  short  time,  you  are  more  likely  to  get 
crystals  of  carbol  fuchsin,  stain  retained  in  deep  scratches  of  the 
glass,  etc.,  all  of  which  a  beginner  may  easily  mistake  for  bacilli. 
The  counterstaining  with  methylene  blue  may  be  shortened  or 
omitted  altogether,  though  this  is  not  advisable,  as  it  is  then  more 
difficult  to  focus  the  film. 

Recognition  of  the  Tubercle  Bacillus. 

The  tubercle  bacillus  is  about  half  as  long  as  a  red  blood- 
corpuscle  is  wide,  or  rather  longer,  and  is  very  slender.  It  is 
straight  or  slightly  curved,  and  is  variable  both  in  shape  and  in 
size  (Plate  II.,  Fig.  2). 

We  recognize  it  by  means  of  a  staining  reaction.  Tubercle 
bacilli  contain  a  considerable  amount  of  fat,  and  this  prevents 
them  from  staining  readily  with  ordinary  stains.  In  the  process 
described  above  we  used  fuchsin,  which  is  a  very  powerful  stain, 
and  added  a  mordant  (carbolic  acid),  which  increases  its  penetrative 
properties.  Even  with  this  staining  is  very  slow,  so  that  we 
heated  the  specimen. 

The  fat  which  prevents  the  bacilli  from  staining  also  prevents 


TUBERCLE  6l 

the  stain  from  being  removed  by  such  substances  as  acids  and 
alcohol.  In  stage  3  of  the  above  process  we  aim  at  allowing  the 
acid  to  act  until  it  has  removed  the  fuchsin  from  everything 
except  the  tubercle  bacilli.  The  methylene  blue  is  a  counter- 
stain,  and  colours  all  organisms,  pus  cells  (especially  their  nuclei), 
epithelial  cells,  and  shreds  of  lung-tissue — in  fact,  everything 
except  the  tubercle  bacilli.  The  latter  appear  as  slender  red  rods, 
which  often  show  the  irregular  staining  which  has  been  described 
as  occurring  in  the  diphtheria  bacillus. 

Now,  "  acid-fast "  bacilli  are  very  rare,  though  they  have  been 
found  in  unexpected  situations  of  late  years.  Only  three  such 
bacilli  need  be  taken  into  consideration  in  dealing  with  human 
pathology.  These  are  the  tubercle  bacillus,  the  leprosy  bacillus, 
and  the  smegma  bacillus.  The  bacillus  of  leprosy  would  rarely 
lead  to  mistakes  in  this  country ;  it  is  recognized  by  the  fact  that 
it  is  straighter  and  more  uniform  than  the  tubercle  bacillus,  and 
by  the  fact  that  it  resists  decolorization  more  powerfully  than  the 
tubercle  bacillus.  The  smegma  bacillus  may  occur  in  the  urine, 
and  lead  to  mistakes  unless  the  sample  examined  was  drawn  off 
per  catheter.  It  is  distinguished  by  the  fact  that  it  is  readily 
decolorized  by  alcohol  (absolute  alcohol  or  methylated  spirit), 
while  the  tubercle  bacillus  is  not.  In  staining  a  film  from  the 
urine,  we  decolorize  in  spirit  for  a  quarter  of  an  hour  after  the 
acid  and  before  the  methylene  blue — i.e.,  between  stages  3  and  4 
in  the  above  description. 

In  searching  for  the  tubercle  bacillus,  the  J-inch  lens  will  serve, 
though  an  oil-immersion  lens  is  an  advantage. 


Method  of  Collecting   the  Sputum. 

This  is  of  some  importance,  and  the  method  recommended 
should  be  carried  out  in  all  cases. 

Get  the  patient  to  wash  out  his  mouth  thoroughly  with  warm 
water  before  going  to  bed.  Let  him  spit  into  a  clean  bottle,  jar 
or  tin,  and  employ  only  the  sputum  coughed  up  before  food  is 
taken  in  the  morning. 

It  is  often  impossible  to  get  sputum  from  children,  but  in  them 
it  is  often  possible  to  pick  masses  of  sputum  out  of  the  vomit  and 
to  demonstrate  tubercle  bacilli  therein ;  and  when  no  such  masses 
are  seen,  bacilli  may  occasionally  be  found  if  the  whole  vomit  be 
carbolized  in  the  manner  described  subsequently  and  the  sedi- 


62  CLINICAL    BACTERIOLOGY   AND    H.EMATOLOGY 

ment  examined.  In  some  cases  in  which  there  is  no  vomit  it  is 
occasionally  justifiable  to  give  an  emetic  for  the  purpose  of 
securing  sputum  and  establishing  the  diagnosis. 


Method  of  Preparation  of  the  Film. 

Sputum. — Pour  the  sputum  into  a  Petri  dish  or  watch-glass, 
and  place  the  vessel  upon  a  dark  surface.  Examine  it  closely, 
looking  out  for  small  yellow  particles ;  these  consist  of  caseous 
material,  and  will  probably  contain  tubercle  bacilli  in  large 
numbers.  The  advantage  of  getting  the  patient  to  wash  out  his 
mouth  and  using  only  fasting  sputum  is  obvious,  for  particles  of 
food  may  present  exactly  the  same  appearance. 

Having  found  such  a  mass,  pick  it  out  by  means  of  a  platinum 
loop  or  pair  of  forceps,  and  transfer  it  to  the  middle  of  a  clean 
slide.  Now  place  another  slide  on  the  top  of  the  first,  squeeze 
them  together,  and  then  slide  them  apart.  You  should  get  two 
good  uniform  films.    Allow  to  dry  spontaneously,  and  fix  by  heat. 

If  there  are  no  caseous  masses,  pick  out  a  mass  of  the  sputum 
at  random  and  proceed  as  before. 

If  no  bacilli  are  found  at  the  first  examination,  and  you  still 
suspect  tubercle,  proceed  as  follows :  Half  fill  an  ordinary 
medicine  bottle  with  carbolic  lotion  (i  in  20),  and  add  a  drachm  or 
two  of  the  sputum.  Shake  thoroughly  for  a  few  minutes,  and 
place  the  bottle  where  you  can  give  it  an  occasional  shake  during 
the  next  few  hours.  Then  pour  the  milky  emulsion  which  results 
into  a  conical  urine-glass,  and  allow  it  to  stand  for  twelve  hours  or 
more.  Remove  some  of  the  deposit  which  will  form  with  a 
pipette,  and  spread  it  into  a  thin  film  on  a  slide.     Dry  and  fix. 

Another  method  which  may  be  used  in  difficult  cases  is  to 
digest  the  sputum  with  pepsin  and  PI  CI— a  pinch  of  the  former 
and  sufficient  of  the  latter  to  make  the  fluid  faintly  acid — in  an 
incubator  for  a  couple  of  hours,  shaking  occasionally,  and  then  to 
boil  the  digested  material  to  prevent  further  action.  The  result 
may  be  centrifugalized  or  allowed  to  deposit,  and  the  sediment 
examined. 

Urine  may  be  centrifugalized  or  allowed  to  stand  without 
previous  addition,  but  better  results  are  obtained  if  carbolic  acid 
(Hquefied  or  in  crystals)  is  added  to  the  urine  in  amount  sufficient 
to  convert  it  into  a  i  in  20  solution.  This  is  allowed  to  deposit  or 
(better)  is  centrifugalized.     Films  are  prepared  from  the  deposit. 


TUBERCLE  63 

Remember  that  they  should  be  left  in  alcohol  for  a  quarter  of 
an  hour  or  so  after  staining. 

Pus  is  best  carbolized  in  the  same  way  as  sputum  ;  if  very 
thin  it  may  be  treated  like  urine.  The  tubercle  bacilli  will 
rarely  be  found  in  pus  unless  it  is  examined  soon  after  the 
abscess  is  opened,  but  may  be  detected  by  inoculation  experi- 
ments for  long  periods. 

Clear  exudates  are  more  difficult  to  examine,  and,  as  they  usually 
contain  bacilli  in  very  small  numbers  only,  a  negative  result 
should  not  be  given  too  much  weight.  (For  the  best  method  to 
employ,  see  p.  121.)  The  examination  is  best  made  in  a  bacterio- 
logical laboratory,  as  decisive  results  can  only  be  obtained  by 
animal  experiments.  Collect  the  f^uid  in  a  bottle  which  has 
previously  been  boiled  in  water  for  half  an  hour  and  allowed 
to  cool.  Cork  it  with  a  cork  which  has  also  been  boiled. 
Add  no  antiseptic,  and  forward  it  to  the  laboratory  as  soon  as 
possible. 

Milk  may  be  examined  in  the  same  way  as  urine,  films  being 
made  from  the  cream  as  well  as  from  the  deposit.  These  films 
are  fixed,  soaked  in  ether  to  remove  fat,  and  again  fixed.  They 
are  then  stained  as  before,  and  it  is  advisable  to  pass  them 
through  alcohol. 

The  results  obtained  by  this  examination  are  somewhat  un- 
certain, as  some  of  the  other  acid-fast  bacilli  found  in  milk  are 
almost  exactly  like  the  tubercle  bacillus,  and  animal  experiments 
are  necessary  for  definite  proof. 

When  fceces  are  to  be  examined,  the  best  plan  is  to  administer 
opium  in  amount  sufficient  to  cause  constipation.  The  surface  of 
the  scybalous  motions  which  result  are  to  be  scraped  off  and 
stained  in  the  usual  way. 

Interpretation  of  Results. 

The  finding  of  tubercle  bacilli  in  the  sputum  is  conclusive 
evidence  of  tuberculosis  of  the  lungs,  but  no  information  as  to 
prognosis  can  be  drawn  from  the  numbers  which  are  present ; 
they  may  occur  in  great  quantities  in  the  sputum  from  patients 
who  are  doing  well,  and  the  author  has  found  enormous  numbers 
in  the  sputum  of  a  person  who  had  presented  no  symptoms  of  the 
disease  for  eight  years,  and  was  apparently  cured.  But  a  person 
in  whom   the  bacilli  are  present  is  always  in   danger  of   a  re- 


64  CLINICAL    BACTERIOLOGY   AND    H.EMATOLOGY 

crudescence  of  the  disease,  a«^  may  he  a  source  of  infection.  Absence 
of  the  bacilli  does  not  disprove  the  diagnosis  of  tuberculosis ; 
bacilli  do  not  appear  in  the  sputum  until  the  lung-tissue  in  which 
they  occur  breaks  down,  and  are  therefore  absent  in  the  early 
stages  of  acute  tuberculosis. 

In  some  cases  of  ordinary  chronic  phthisis  bacilli  may  occur  in 
the  sputum  in  very  scanty  numbers,  and  may  be  missed  unless  a 
very  careful  search  is  made.  Bacilli  should  not  be  considered  as 
being  absent  until  well-stained  films  have  been  examined  for 
at  least  half  an  hour,  and  the  examination  repeated  on  several 
occasions.  It  is  in  these  cases  that  the  carbolic  method  of 
examination  is  so  useful. 

The  finding  of  tubercle  bacilli  in  the  urine  is  practically  abso- 
lute proof  of  tuberculosis  of  some  part  of  the  urinary  tract, 
probably  the  kidneys  or  bladder.  Absence  of  bacilli  implies 
nothing  unless  the  examination  has  been  made  very  thoroughly 
and  repeated  several  times  at  intervals.  Then  it  affords  pve- 
sumptive  evidence  that  the  urinary  passages  are  free  from  the 
disease. 

The  same  is  true  of  the  examination  of  pus.  Tubercle  bacilli 
rarely  occur  in  inflammatory  exudates  except  in  very  small 
numbers,  and  can  often  only  be  demonstrated  by  animal  experi- 
ments. If  you  examine  pus  from  a  chronic  abscess  and  find  no 
organisms  of  any  kind,  it  is  almost  certain  that  the  process  is  a 
tuberculous  one,  and  the  negative  evidence  obtained  by  the  failure 
to  find  tubercle  bacilli  should  not  be  allowed  to  carry  much 
weight.     The  same  is  true  for  the  clear  exudates. 

In  suspected  cases  of  tuberculosis,  in  which  no  infective 
material  is  forthcoming,  there  are  two  methods  which  may  be 
used  :  (i)  The  estimation  of  the  opsonic  index  (see  p.  148),  and 
(2)  the  use  of  Koch's  old  tuberculin.  This  is  usually  considered  to 
be  too  dangerous  for  practical  use,  but  if  a  small  dose  only  be  given 
(tu^  c.c),  and  if  it  is  not  employed  on  cases  in  which  there  is  a 
mixed  infection,  I  believe  the  danger  to  be  practically  nil.  The 
tuberculin  is  sold  in  i-c.c.  bottles.  For  use,  add  the  contents  of 
one  of  these  to  200  c.c.  (about  gvii.)  of  recently  boiled  and  cooled 
normal  saline  solution.  Mix  well  and  inject  i  c.c.  (ni^xvii.)  of 
the  fluid  hypodermically.  In  a  positive  case  there  will  be  a  sharp 
reaction,  the  temperature  rising  to  103°  or  more,  and  remaining 
elevated  for  a  day  or  so. 

Information  may  also  be  obtained  in  some  cases  by  a  blood- 


LEPROSY  65 

count  or  by  cyto-diagnosis  (see  pp.  227  and  237).  Calmette's 
ophthalmo -reaction  may  also  be  used,  but  is  by  no  means  devoid  of 
danger. 

LEPROSY  ^ 

The  leprosy  bacillus  resembles  that  of  tubercle,  but  it  is 
somewhat  straighter  and  more  uniform.  It  occurs  in  leprous 
lesions  ip  great  profusion,  and  its  discovery  does  not  present  any 
difficulty.    It  has  never  been  cultivated. 

In  a  suspected  case  of  leprosy  films  should  be  made  from  the 
nasal  discharge,  for  the  nasal  cavities  are  very  frequently  affected. 
Indeed,  it  seems  highly  probable  that  the  primary  lesion  through 
which  the  bacilli  gain  access  to  the  body  is  in  the  nose  in  most 
cases.  A  small  portion  of  one  of  the  leprous  nodules  may  also  be 
removed,  and  films  made  by  rubbing  the  cut  surface  against  a 
clean  slide.  If  there  is  an  ulcer,  films  may  be  made  from  the 
secretion  from  it. 

Films  should  be  stained  by  the  method  which  we  have  recom- 
mended for  the  tubercle  bacillus.  If  bacilli  are  present  in  large 
quantities,  the  case  is  almost  certainly  one  of  leprosy,  for  tubercle 
bacilli  are  never  found  in  similar  situations  except  in  scanty 
numbers.  If  a  doubt  should  arise  as  to  the  identity  of  the 
bacilli,  advantage  should  be  taken  of  the  fact  that  the  leprosy 
bacillus  retains  the  fuchsin  even  more  firmly  than  the  tubercle 
bacillus  when  exposed  to  the  action  of  an  acid.  A  film  from  the 
suspected  material  should  be  spread  at  one  end  of  the  slide,  and 
some  sputum  known  to  be  rich  in  tubercle  bacilli  at  the  other ; 
the  whole  should  be  stained  by  hot  carbol  fuchsin,  and  decolorized 
by  being  immersed  bodily  in  25  per  cent,  sulphuric  acid  for  half 
an  hour.  If  the  tubercle  bacilli  are  decolorized,  any  bacilli  which 
have  retained  the  red  colour  are  almost  certainly  those  of  leprosy. 
If  the  tubercle  bacilli  are  not  decolorized,  a  fresh  specimen  should 
be  prepared  and  immersed  in  the  acid  for  a  longer  period. 

ACTINOMYCOSIS,  OR  STREPTOTHRICOSIS 

Actinomycosis  is  very  closely  aUied  to  tuberculosis  ;  the  lesions 
appropriate  to  the  two  diseases  are  almost  identical  in  histological 
appearance,  and  the  granuloma  which  occurs  in  actinomycosis 
goes  on  to  fibrosis  or  to  the  formation  of  "  cold  abscesses  "  just  as 
a  tubercle   may   do.     The   formation  of  fibrous   tissue  is  most 

5 


66  CLINICAL    BACTERIOLOGY   AND    HEMATOLOGY 

marked  in  cattle,  and  in  them  the  disease  is  more  chronic  ; 
suppuration  is  more  common  in  man,  and  the  disease  runs  a  more 
rapid  course. 

The  pus  from  an  actinomycotic  abscess  is  often  viscid,  and 
contains  a  greater  or  smaller  number  of  small  greenish,  yellow,  or 
brownish  nodules.  They  are  about  as  large  as  the  head  of  a  very 
small  pin,  and  are  quite  opaque ;  under  the  low  power  of  the 
microscope  such  a  granule  has  a  coarsely  granular  appearance, 
and  looks  something  like  a  raspberry.  If  nodules  presenting 
these  appearances  are  found  in  any  specimen  of  pus,  whatever 
be  its  origin,  a  careful  microscopic  examination  should  be  made 
to  determine  its  nature.     This  is  not  difficult. 

Method. — Place  some  of  the  pus  which  contains  these  granules 
on  a  clean  slide,  and  press  another  slide  upon  it  so  as  to  crush 
the  granules  ;  dry,  fix,  and  stain  by  Gram's  method.  Do  not 
counter-stain. 

Tumours  removed  or  incised  at  an  operation,  or  organs  removed 
at  a  post-mortem  examination,  should  have  their  cut  surfaces 
rubbed  upon  the  surface  of  a  slide,  and  the  film  thus  obtained 
treated  in  a  similar  way ;  or  they  may  be  scraped,  and  the 
scrapings  spread  on  a  slide.  Sections  may  also  be  cut,  but  are 
not  usually  necessary  for  the  diagnosis. 

Examination  of  the  Specimens. 

Actinomycosis  of  cattle  is  caused  by  the  ray  fungus,  an 
organism  which  derives  its  name  from  the  star-shaped  colonies 
which  it  forms  whilst  growing  in  the  tissues.  It  consists  of  two 
chief  parts ;  the  central  portion  of  the  colony  is  formed  of  a 
network  of  narrow  filaments,  which  have  a  radial  arrangement 
at  the  periphery  (Plate  II.,  Fig.  6).  In  this  part  small  bodies 
which  have  the  appearance  of  cocci  may  often  be  seen.  The 
outer  zone  consists  of  the  clubs  which  (when  present)  are  so 
characteristic.  These  clubs  are  flask-shaped  expansions  of  the 
sheath  of  the  radial  filaments  already  mentioned,  and  are  arranged 
with  their  narrow  extremities  pointing  inwards.  They  are  not 
generally  present  in  man,  and  when  present  are  often  badly 
developed ;  they  are  much  more  common  and  more  perfect  in  the 
ox,  where  the  disease  is  more  chronic  and  pus-formation  rare. 

The  reason  for  this  is,  perhaps,  partly  that  man  is  less  resistant 
against  the  organism,  but  probably  the  chief  factor  is  the  difference 


ACTINOMYCOSIS,    OR   STREPTOTHRICOSIS  67 

in  the  fungus  present.  The  organism  in  bovine  actinomycosis  is 
the  ray  fungus,  which  is  described  above  ;  but  in  man  there  are 
numerous  species  of  fungus  which  can  bring  about  infection  and 
cause  the  disease  known  cUnically  as  actinomycosis :  this  disease 
is  therefore  not  a  specific  entity,  due  to  a  single  cause,  but  a 
group  of  alHed  diseases,  just  as  suppuration  is.  The  organisms 
in  question  are  all  members  of  the  genus  Streptothrix,  and  consist 
of  long  filaments  of  mycehum,  which  differ  from  the  bacteria  in 
showing  true  branching  and  in  breaking  up  into  "  chain  spores," 
resembling  chains  of  cocci.  The  different  species  vary  very 
greatly  in  cultural  characters,  but  there  are  also  marked 
differences  in  the  appearances  met  with  in  films  of  pus,  etc.  In 
what  may  be  regarded  as  typical  cases,  the  nodules  described 
above,  when  flattened  out  and  stained  by  Gram's  method,  show  a 
central  portion  consisting  of  a  tangled  mass  of  narrow  mycelial 
threads,  some  of  which  may  show  the  degeneration  into  chain- 
spores,  and  look  like  streptococci,  whilst  there  ..may  or  may  not 
be  a  peripheral  portion  showing  a  radial  arrangement  (Plate  II., 
Fig.  6,  which  was  drawn  from  a  remarkably  perfect  specimen). 
These  colonies  vary  greatly  in  size,  but  do  not  usually  fill  up 
more  than  half  the  field  of  an  oil-immersion  lens,  so  that  it  is  best 
to  search  for  them  in  Gram  specimens  (not  counterstained)  with  a 
low  power,  and  to  turn  successively  the  ^  and  ^^  on  to  any  small 
violet  masses  which  may  be  seen.  In  other  cases  the  fungus  will 
assume  the  form  of  threads  in  masses  without  any  definite 
arrangement,  and  in  others  the  threads  will  be  isolated ;  in  either 
case  it  will  usually  be  possible  to  find  threads  showing  true 
branching  or  chain  spores,  and  this  is  sufficient  for  the  diagnosis. 
In  yet  others  the  bulk  of  the  mycelium  splits  up  into  short  lengths 
greatly  resembling  bacilli,  and  when  this  happens  the  diagnosis 
may  be  missed  unless  a  careful  search  happens  to  reveal  an 
unbroken  piece  of  mycelium  (Plate  IV.,  Fig.  i). 

Cultures  are  usually  difficult  to  prepare,  and  are  not  much  help 
in  the  diagnosis. 

The  importance  of  making  this  examination  as  a  routine  method 
in  all  cases  in  which  the  diagnosis  is  not  absolutely  clear  must  be 
strongly  urged  on  all  practitioners,  since  an  accurate  diagnosis  of 
actinomycosis  may  be  followed  in  many  cases  by  a  cure  by  means 
of  large  doses  of  iodide  of  potassium.  Actinomycosis  has  a  habit 
of  turning  up  when  least  expected ;  thus,  I  have  found  it 
accidentally  in  three  cases  in  the  sputum  where  the  true  diagnosis 

5—2 


68  CLINICAL    BACTERIOLOGY   AND    HEMATOLOGY 

was  not  suspected ;  once  in  an  enlarged  tonsil ;  once  in  an  appar- 
ently typical  case  of  cancer  of  the  breast,  etc. 

The  films  should  be  carefully  examined  for  the  presence  of 
these  structures.  Clubs  are  not  likely  to  be  found  in  the  pus, 
and  their  absence  does  not  tell  against  the  diagnosis ;  the  dense 
felted  network  of  filaments  retaining  Gram's  stain  is  what  is  to  be 
looked  for,  and  its  presence  is  quite  sufficient  for  a  diagnosis. 
Fortunate  specimens  may  show  a  complete  colony,  with  its 
irregular  network  in  the  centre  and  the  radial  arrangement  of  the 
fibres  on  the  periphery,  or  there  may  be  mere  fragments  of 
mycelium. 

GLANDERS 

Glanders  is  one  of  the  infective  granulomata,  and  is  closely 
allied  to  tuberculosis ;  it  differs,  however,  in  running  a  more  rapid 
course  and  in  the  greater  tendency  which  the  specific  lesions 
exhibit  to  undergo  suppuration.  It  is  caused  by  the  B.  mallei, 
an  organism  which  is  nearly  as  long  as  the  tubercle  bacillus, 
and  decidedly  thicker.  It  stains  readily  with  all  stains,  and  is 
easily  decolorized ;  it  loses  its  stain  when  treated  by  Gram's 
method,  and  does  not  form  spores. 

The  bacteriological  diagnosis  of  the  disease  is  not  easy,  and 
should  be  referred  to  a  bacteriologist.  A  quantity  of  the  discharge 
from  a  suspected  case  should  be  taken  with  aseptic  precautions, 
and  transmitted  as  soon  as  possible  in  a  test-tube  or  bottle  which 
has  been  sterilized  by  dry  heat  or  by  boiling.  Pus  had  better  be 
sent  in  a  pipette. 

Where  abscesses  are  opened,  cultures  taken  direct  from  the  pus 
may  possibly  contain  the  bacillus  in  pure  culture.  In  this  case  it 
may  be  identified  by  the  characters  of  its  growth  upon  potato. 
The  colonies  have  the  colour  and  appearance  of  honey  at  first ; 
they  grow  very  rapidly,  coalesce,  and  the  potato  is  soon  covered 
with  a  moist-looking  film,  which  afterwards  becomes  brown,  the 
surface  of  the  medium  in  the  neighbourhood  becoming  greenish- 
brown.  If  cultures  from  pus  grown  on  potato  exhibit  these 
appearances,  and  contain  a  short  and  thick  bacillus  which  does 
not  stain  by  Gram's  method,  the  case  may  be  diagnosed  as  being 
probably  one  of  glanders,  even  although  the  culture  be  not  a 
pure  one. 


TYPHOID   FEVER  69 


TYPHOID  FEVER 

Typhoid  fever  is  caused  by  a  bacillus  which  is  variable  in 
length,  though  usually  short  (about  half  as  long  as  a  tubercle 
bacillus)  and  thick,  its  length  being  only  about  three  times  its 
breadth  ;  very  long  forms  also  occur,  but  in  small  numbers.  It 
does  not  form  spores,  and  it  does  not  stain  by  Gram's  method. 
It  is  actively  motile ;  when  a  culture  of  the  organism  in  a  fluid 
medium  is  examined  under  the  microscope,  the  bacilli  can  be 
seen  darting  rapidly  about  in  all  directions.  The  bacillus  owes 
its  motility  to  the  possession  of  a  large  number  of  long,  wavy 
flagella,  which  can  only  be  seen  after  special  and  difficult  staining 
processes. 

The  B.  coli  communis,  the  most  plentiful  organism  of  the  in- 
testine in  man  and  animals,  bears  a  very  close  resemblance  to 
the  typhoid  bacillus,  and  can  only  be  distinguished  therefrom  by 
the  application  of  several  cultural  and  chemical  tests,  the  perform- 
ance of  which  takes  a  considerable  amount  of  time.  This  renders 
it  very  difficult  to  diagnose  typhoid  fever  by  methods  similar  to 
those  which  are  in  use  for  the  other  diseases  mentioned — i.e.,  by 
the  demonstration  of  the  specific  organism.  Suppose,  for  instance, 
that  we  were  to  attempt  to  determine  the  nature  of  a  case  of 
diarrhcea  by  a  search  for  the  typhoid  bacillus  in  the  stools.  For 
every  typhoid  bacillus  which  we  should  encounter  we  should  find 
a  great  many  colon  bacilli,  and  we  should  only  be  able  to  dis- 
tinguish the  one  from  the  other  by  a  prolonged  and  careful 
examination  of  pure  cultures.  It  is  quite  certain  that  the  disease 
might  be  diagnosed  in  this  way ;  indeed,  it  has  been  done,  but  the 
task  is  an  extremely  difficult  one,  and  the  diagnosis  would  be 
delayed  for  a  considerable  period.* 

In  other  regions  in  which  the  typhoid  bacillus  occurs  during  an 
attack  of  typhoid  fever  the  search  is  usually  facilitated  by  the 
absence  of  other  organisms,  especially  by  the  absence  of  the 
B.  coli.  The  specific  bacillus  occurs  in  the  blood,  spleen,  spots, 
mesenteric  glands,  liver,  and  frequently  in  the  urine. 

It  may  often  be  demonstrated  in  the  Mood,  and  the  method  is 
now  acquiring  some  importance  from  the  fact  that  positive  results 
are  found  to  be  very  frequent  if  the  examination  is  made  early  in 

*  Modern  methods  have  greatly  faciUtated  the  task,  but  even  now  it  is  only- 
used  when  other  methods  are  unavailable,  or  for  certain  special  purposes. 


70  CLINICAL    BACTERIOLOGY   AND    HEMATOLOGY 

the  disease — i.e.,  within  the  first  week.  Thus  it  is  available  before 
the  Widal  reaction  appears,  and  should  be  used  wherever  a 
positive  diagnosis  is  required  at  a  very  early  date.  The  blood  is 
drawn  direct  from  a  vein  by  one  of  the  methods  described  on 
p.  144,  and  at  least  5  c.c.  should  be  taken,  whilst  10  c.c.  gives  a 
greater  probability  of  a  successful  result.  The  best  culture 
medium  to  employ  is  broth  to  which  -5  to  i  per  cent,  of  sodium 
citrate  has  been  added ;  this  prevents  the  coagulation  of  the 
blood  and  the  consequent  entanglement  of  the  bacilli  in  the  clot, 
which,  when  it  occurs,  greatly  delays  the  appearance  of  the 
growth.  The  amount  of  blood  indicated  above  should  be 
inoculated  into  100  c.c.  of  citrated  broth  and  incubated  at  37°  C. 
Growth  will  probably  be  seen  in  twenty-four  hours  or  less.  Un- 
fortunately, typhoid  bacilli  fresh  from  the  body  often  fail  to 
agglutinate  with  typhoid  serum,  and  only  acquire  that  property 
after  cultivation  for  several  generations  on  artificial  media  :  this, 
the  best  test,  is  not  always  conclusive.  But  all  cases  clinically 
resembling  early  typhoid  which  give  a  culture  of  motile,  non- 
sporulating.  Gram-negative  bacilli  of  the  morphological  characters 
described  above,  should  be  regarded  as  early  typhoid,  and  in  all 
probability  the  appearance  of  the  Widal  reaction  will  soon  settle 
the  matter.  The  other  possible  diagnoses  are  extremely  unlikely. 
The  further  identification  of  the  bacillus  is  a  matter  of  some 
difficulty  for  which  expert  help  should  be  obtained,  or  one  of  the 
larger  manuals  of  bacteriology  consulted. 

The  mesenteric  glands  and  liver  are,  of  course,  not  available 
or  the  purpose  of  diagnosis. 

The  demonstration  of  the  bacilli  in  the  urine  is  sometimes  quite 
easy,  and  might  be  of  some  diagnostic  value.  But  they  do  not 
always  occur  in  this  excretion,  and  the  B.  coli  often  does  so,  and  it 
is  not  easy  to  distinguish  between  the  two. 

The  only  way  in  which  typhoid  fever  can  be  diagnosed  with 
ease  and  certainty  by  a  demonstration  of  the  specific  bacillus  is 
by  an  examination  of  material  drawn  directly  from  the  spleen  by 
means  of  a  hypodermic  needle.  The  organism  occurs  constantly  in 
this  situation,  and  its  demonstration  is  not  difficult.  The  necessary 
operation,  however,  is  by  no  means  devoid  of  risk,  and  is  now 
generally  abandoned. 

This  brings  us  to  the  method  in  which  typhoid  fever  is  now 
usually  diagnosed  by  the  bacteriologist — Widal's  reaction.     This 


TYPHOID    FEVER  7I 

reaction  is  a  special  example  of  a  general  law  which  was  discovered 
by  Durham  and  others,  and  which  is  to  the  effect  that  the  blood- 
serum  of  a  person  who  has  been  through  an  attack  of  a  bacterial 
disease  will  cause  the  specific  organism  of  that  disease  to  collect 
into  clumps.  For  instance,  if  we  take  a  broth  culture  of  the  vibrio 
of  Asiatic  cholera  (which  is  turbid  and  opalescent)  and  add  to  it  a 
small  quantity  of  blcod-serum  from  a  patient  who  has  recovered 
from  an  attack  of  cholera,  we  shall  find  that  the  culture  becomes 
clear,  a  sediment  collecting  at  the  bottom  of  the  tube  ;  and  if  we 
examine  this  sediment  we  shall  find  that  it  consists  of  felted 
masses  of  the  vibrios.  This  reaction  is  a  general  one,  and  is 
given  in  most,  if  not  all,  bacterial  diseases.  But  Widal,  Griin- 
baum,  and  others,  working  independently  about  the  same  time, 
showed  that,  whereas  in  many  diseases  it  is  a  reaction  of  immunity 
{i.e.,  does  not  occur  until  late  in  or  after  the  disease),  in  typhoid 
fever  it  is  a  reaction  of  infection,  and  occurs  so  early  in  the  course 
of  the  disease  that  it  is  of  great  value  in  diagnosis. 

The  test  is  applied  by  adding  a  small  quantity  of  the  serum 
from  the  patient  suspected  to  be  suffering  from  the  disease  to  a 
larger  amount  of  a  young  culture  of  typhoid  bacilli,  and  watching 
whether  the  appearance  of  the  culture  undergoes  any  change  :  it 
may  be  watched  under  the  microscope  or  by  the  naked  eye,  the 
technique  differing  in  the  two  cases.  The  microscopic  method  is 
rapid  and  requires  a  very  small  amount  of  blood,  and  is  now 
generally  used.  The  macroscopic  method  is  perhaps  somewhat  easier 
for  a  beginner,  but  it  takes  a  longer  time  and  requires  a  larger 
amount  of  blood-serum. 

The  technique  in  either  case  is  readily  learnt,  and  can  be 
carried  out  with  very  little  difficulty  if  the  materials  are  at  hand. 
But  the  test  is  one  which  it  is  seldom  advisable  for  the  home 
worker  to  attempt,  as  it  requires  a  young  culture  of  typhoid 
bacilli.  We  shall,  however,  describe  the  process,  as  some  may 
carry  it  out  during  an  epidemic,  or  if  they  are  living  where  they 
have  not  access  to  a  public  laboratory.  The  process  has  now 
been  greatly  facilitated  by  the  demonstration  of  the  fact  (by 
Widal)  that  the  reaction  is  given  with  dead  cultures  of  the  bacilli. 
These  cultures  can  be  obtained  from  any  bacteriological  laboratory, 
and  will  keep.  They  are  prepared  by  adding  4  or  5  c.c.  of 
the  following  fluid  to  an  eighteen-hours-old  culture  of  typhoid 
bacilli,  scraping   off  the  growth,  pipetting  the  emulsion  into  a 


72  CLINICAL    BACTERIOLOGY   AND    HEMATOLOGY 

Sterile  test-tube,  and  heating  to  60°  C.  for  half  an-hour  to  ensure 
sterility : 

Normal  saline  solution  (o*8  per  cent.)  -       450  c.c. 

Carbolic  acid  (liquefied)       -  -  -  1-25  c.c. 

Glycerin      -  -  -  -  -         ^o  c.c. 

Boil  and  allow  to  cool  immediately  before  use.* 

The  bacilli  gradually  separate  from  an  emulsion  prepared  in 
this  way,  but  are  distributed  throughout  the  fluid  by  shaking. 
With  a  supply  of  this  culture  at  hand  the  practitioner  can  apply 
the  test  at  home  without  difficulty. 

Collect  the  blood  from  the  ear,  as  described  on  p.  34,  taking 
care  to  get  a  Wright's  pipette  about  half  full.  The  test  may  be 
applied  to  a  dry  drop  of  blood,  but  in  this  case  there  is  no 
possibility  of  making  an  accurate  dilution,  and  unless  a  fair 
quantity  of  serum  is  at  hand  it  is  impossible  to  perform  the  test 
by  the  macroscopic  method. 

If  a  pipette  is  not  at  hand,  it  is  easy  to  manufacture  one  out 
of  any  piece  of  glass  tubing  which  may  be  available. 

Method  of  performing  Widal's  Reaction  by  the 
Microscopic  Method. 

Requisites. — i.  A  young  culture  (not  more  than  eighteen  hours 
old)  of  typhoid  bacilli  on  agar. 

Where  dead  bacilli  are  to  be  used,  this  is  to  be  replaced  by  a 
culture  obtained  from  the  laboratory  and  prepared  in  the  manner 
described. 

2.  A  small  funnel  provided  with  a  double  thickness  of  white 
filter-paper.     This  is  unnecessary  if  dead  cultures  are  to  be  used. 

3.  Three  clean  watch-glasses. 

4.  A  platinum  loop.  This  should  be  made  of  fine  wire  and 
have  a  loop  (which  must  be  completely  closed)  about  j^^  inch  in 
diameter. 

5.  A  hollow-ground  slide.  This  is  an  ordinary  slide  having  a 
well  about  \  inch  in  diameter  sunk  in  its  centre.  If  it  is 
not  at  hand  a  cell  may  be  built  up  on  an  ordinary  slide.  Take  a 
piece  of  thin  card  i  inch  square  and  cut  out  a  square  \  inch  in 
diameter  from  its  centre.  Fix  this  perforated  square  down  on  to 
the  slide  with  vaseline  or  immersion  oil. 

*  J.  H.  Borden,  Proc.  N.  Y.  Path.  Soc,  1904. 


TYPHOID    FEVER 


73 


6.  Thin  cover -glasses. 

7.  The  microscope.  The  test  can  be  carried  out  quite  well 
with  a  J-inch  lens. 

Process — i.  Making  the  Emulsion.— Vom  a  small  quantity  of 
tap-water  into  the  culture-tube,  or,  better,  scrape  off  some  of  the 
growth  and  mix  it  with  some  water  in  a  watch-glass.  In  either 
case  stir  it  round  with  the  platinum  needle  for  a  few  minutes,  so 
that  the  bacilli  are  evenly  distributed  throughout  the  water  and 
form  an  emulsion. 

Next  take  the  hollow-ground  slide  and  paint  a  ring  of  immersion 
oil  round  the  well  (Fig.  22,  h).     If  you  are  using  a  built-up  cell, 


Fig,  22. —Hanging-drop  Preparation.     (Crookshank.) 

paint  the  top  of  the  card  with  the  oil.  In  either  case  vaseline 
may  be  used. 

Place  a  drop  of  the  emulsion  on  a  clean  dry  cover-glass,  and 
invert  the  hollow-ground  slide  over  it ;  press  it  down  so  that  the 
oil  round  the  well  adheres  to  the  cover-glass ;  now  invert  the 
slide,  and  you  will  have  a  hanging-drop  specimen.  The  bacilli  will 
be  contained  in  the  droplet  of  water  (Fig.  22,  a)  which  hangs 
from  the  lower  surface  of  the  cover-glass  :  this  will  not  dry  it  up 
if  the  seal  made  by  the  oil  is  perfect. 

Place  the  specimen  under  the  microscope  and  examine  it  with 
the  low  power,  using  the  fine  adjustment  and  stopping  down 
the  diaphragm.  Focus  until  the  surface  of  the  cover-glass  is 
distinctly  seen,  and  then  move  the  slide  about  until  the  edge  of 
the  hanging-drop  runs  across  the  centre  of  the  field.  Then  turn 
on  the  i-inch  lens  and  open  and  shut  the  diaphragm  until  the 
field  is  faintly  illuminated ;  the  exact  amount  of  light  required 
can  only  be  learnt  by  experience. 

Now  focus  up  and  down  very  carefully,  using  the  fine  adjustment, 


74  CLINICAL    BACTERIOLOGY   AND    HEMATOLOGY 

until  you  see  a  line  running  across  the  field  and  dividing  it  into 
a  lighter  and  a  slightly  darker  portion.  This  is  the  edge  of  the 
drop.  Focus  a  little  deeper  ;  you  should  see  numerous  small 
unstained  bacilli,  and  if  these  are  not  visible  it  probably  indicates 
that  the  illumination  is  not  right.  Open  and  shut  the  diaphragm, 
keeping  a  sharp  look-out  down  the  microscope  all  the  time.  It 
may  help  matters  to  lower  the  condenser  for  a  short  distance. 

Having  obtained  a  clear  view  of  the  bacilli,  examine  them  for 
motility  and  absence  of  clumps,  and  see  whether  they  are  present 
in  proper  proportion  to  the  amount  of  fluid. 

If  the  culture  is  in  good  condition,  the  bacilli  should  be  seen 
darting  about  in  all  directions ;  but  if  the  movement  is  but 
sluggish,  the  reaction  may  still  be  obtained.  If  the  specimen  is 
kept  for  a  short  time  in  a  warm  place  or  in  the  incubator,  the 
movements  will  usually  become  more  rapid.  It  is  hardly 
necessary  to  say  that  when  dead  cultures  are  used  there  will  be 
no  movements  of  translation,  though  the  bacilli  may  show 
oscillatory  (Brownian)  movements. 

The  specimen  must  be  searched  thoroughly  for  clumps  of 
bacilli,  and  if  these  are  present  the  emulsion  must  be  filtered 
through  a  double  thickness  of  white  filter-paper.  This  examina- 
tion for  clumps  is  a  most  important  part  of  the  process,  and  must 
be  attended  to  whether  dead  or  living  cultures  are  in  use. 

Next  see  that  the  emulsion  is  neither  too  thin  nor  too  thick. 
No  definite  rules  can  be  given,  but  if  there  are  very  few  bacilli 
in  the  field  a  further  supply  of  growth  must  be  added  to  the  stock 
of  emulsion,  and  a  further  specimen  examined.  If  the  bacilli  are 
thickly  crowded  together,  the  emulsion  must  be  diluted  with  a 
little  water  and  re-examined. 

When  you  are  satisfied  that  the  emulsion  is  right,  slide  off  the 
cover-glass  and  drop  it  into  some  antiseptic  lotion  ;  of  course,  this 
is  unnecessary  if  dead  cultures  are  used. 

2.  Making  the  Dilution. — You  are  now  about  to  dilute  a  drop  of 
the  serum  from  the  patient  with  a  known  multiple  (in  this  case 
thirty  times)  of  its  bulk  of  the  emulsion  which  you  have  just 
prepared.  To  do  so  you  will  take  advantage  of  the  fact  that  the 
platinum  loop,  if  dipped  into  a  fluid  and  pressed  against  a  surface, 
so  that  every  part  of  the  loop  touches  that  surface,  will  deposit  a 
drop  of  fluid  of  definite  size.  You  are  about  to  mix  one  loopful 
of  the  serum  with  twenty-nine  loopfuls  of  the  emulsion  just 
prepared  and  examined. 


TYPHOID    FEVER 


75 


Blow  the  blood  from  the  pipette  out  on  to  a  watch-glass  (to  do 
this  it  will  be  necessary  to  break  the  tip  of  the  pipette),  and  tilt 
the  latter  so  that  the  serum  flows  away  from  the  coagulum.  Now 
take  a  loopful  of  the  serum  and  place  it  on  another  watch-glass, 
taking  care  to  put  the  loop  flat  on  the  surface  of  the  glass  ;  this 
is  done  more  easily  if  the  wire  is  slightly  bent,  or  if  a  flat  side  is 
used  instead  of  the  watch-glass. 

Next  heat  the  platinum  loop  in  the  flame;  this  is  to  burn  off  any 
blood  which  might  remain  on  it  and  contaminate  the  emulsion. 
Take  up  a  loopful  of  the  emulsion,  and  place  it  on  the  watch-glass 
by  the  side  of  the  drop  of  serum,  but  not  touching  it.  Repeat  this 
until  you  have  placed  twenty-nine  drops  of  emulsion  round  the 


Fig.  23. 
a,  Negative  Widal's  reaction  ;  b,  positive  Widal's  reaction. 


serum.  Mix  the  whole  together  by  stirring  them  thoroughly  with 
the  platinum  loop,  place  a  droplet  of  the  mixture  on  a  clean  cover- 
glass,  making  a  hanging-drop  specimen,  and  examine  as  before. 

If  the  blood  comes  from  a  case  of  typhoid  fever  (with  certain 
restrictions  which  will  be  discussed  below),  the  microscopic 
appearances  will  be  quite  diflerent  from  those  seen  in  the  drop  of 
emulsion  which  was  previously  examined.  The  bacilli  will  no 
longer  swim  about  rapidly  in  all  directions  ;  'they  will  become 
paralyzed,  and  remain  quite  motionless.  Further,  they  will  collect 
into  clumps,  each  clump  consisting  of  a  larger  or  smaller  number 
of  bacilli,  arranged  in  a  felted  network,  resembling  that  seen  in 
a  heap  of  "  spellicans  "  (Fig.  23,  b).  This  is  the  complete  positive 
reaction  ;  it  consists  of  two  parts,  clumping  and  paralysis,  and  is 
given  only  (in  the  dilution  used)  by  the  blood  of  a  patient  who  is 
suffering  or  who  has  sufl'ered  from  typhoid  fever.  If  this  is  not 
the  case,  the  bacilli  will  continue  to  move  about  just  as  before, 
and  will  not  collect  into  clumps. 


76  CLINICAL    BACTERIOLOGY   AND    H.EMATOLOGY 

In  the  process  which  has  been  described  above,  the  blood  has 
been  diluted  to  thirty  times  its  volume,  and  this  is  the  best  dilution 
to  use  for  diagnostic  purposes.  But  the  reaction  is  given  earlier 
if  a  lesser  dilution  (one  in  ten)  is  used,  though  there  is  then  a 
greater  chance  of  fallacy. 

Sometimes  the  reaction  takes  place  almost  as  soon  as  the  serum 
is  added.  At  other  times  it  is  delayed,  and  for  these  it  is  necessary 
to  fix  a  time  limit.  With  a  dilution  of  i  in  30,  one  hour  is  a 
safe  time-limit  to  adopt,  and  if  the  reaction  takes  place  after 
this  the  result  should  be  looked  upon  with  great  suspicion,  and 
the  test  reapplied  after  a  day  or  two. 

Exactly  similar  processes  are  carried  out  if  dead  cultures  of 
typhoid  bacilli  are  used,  but  here  the  emulsion  should  be  sent  out 
from  the  laboratory  free  from  clumps,  and  containing  exactly  the 
right  number  of  bacilli,  so  as  to  be  ready  for  immediate  use.  But 
the  practitioner  is  urged  not  to  trust  to  such  an  emulsion  without 
making  a  hanging-drop,  and  examining  it  just  before  making  the 
test. 

If  dead  cultures  are  used,  it  is  advisable  to  use  a  rather  less 
degree  of  dilution  than  in  the  above  process.  A  dilution  of  i  in 
20  will  answer  perfectly.     The  time-limit  is  the  same. 

Interpretation  of  Results. 

A  positive  result  may  mean  : 

1.  That  the  patient  is  suffering  from  typhoid  fever. 

2.  That  he  has  suffered  from  typhoid  fever  within  a  certain 
period  before  the  blood  was  taken.  The  hypothetical  substance 
which  we  believe  to  be  the  cause  of  the  reaction  (agglutinin)  con- 
tinues to  be  formed,  or  remains  in  the  blood,  for  some  time  after 
complete  convalescence  from  typhoid  fever;  the  reaction  has 
been  known  to  persist  for  seven  or  eight  years,  and  probably 
usually  does  so  for  about  two.  This  fact  must  be  remembered 
in  interpreting  the  results  obtained  from  Widal's  test.  If  the 
patient  has  suffered  from  typhoid  fever,  or  from  an  obscure 
illness,  which  might  possibly  have  been  typhoid  fever,  a  year  or 
two  previously,  the  positive  reaction  should  be  regarded  with 
suspicion. 

In  such  cases  the  test  should  be  carried  out  so  that  the  smallest 
dilution  which  will  cause  clumping  can  be  ascertained,  and  the 
test  repeated  in  two  or  three  days.     If,  for  instance,  we  found 


m 


TYPHOID    FEVER  77 


at  the  blood  clumps  only  in  a  dilution  of  i  in  20  on  one 
day,  and  in  a  dilution  of  i  in  100  three  days  later,  this  affords 
a  certain  proof  that  the  reaction  is  due  to  a  present  attack 
of  typhoid  fever,  and  is  not  due  to  one  which  took  place  at  a 
previous  date.  This  investigation  should  be  entrusted  to  an 
expert  bacteriologist,  and  plenty  of  blood  sent  on  each  occasion. 

A  negative  result  may  mean  : 

1.  That  the  patient  is  not  suffering  from  typhoid  fever. 

2.  That  he  is  suffering  from  typhoid  fever,  but  the  date  is  too 
early  for  the  appearance  of  the  reaction.  The  reaction  sometimes 
occurs  on  the  fifth  or  sixth  day,  usually  after  the  tenth  day,  and 
in  all  but  a  very  small  number  of  cases  before  the  end  of  the 
second  week.  If  the  onset  of  the  disease  (as  far  as  it  can  be 
fixed)  is  less  than  this,  the  examination  should  be  repeated  after 
two  or  three  days. 

3.  In  a  very  small  number  of  cases  the  reaction  is  delayed  still 
further,  and  if  the  patient  dies  may  not  occur  at  all.  These  cases 
are  usually  severe  ones,  and  do  not  present  any  difficulty  in 
diagnosis.  Sometimes  the  reaction  is  delayed  well  into  the  con- 
valescence in  mild  attacks,  but  this  is  exceedingly  rare. 

Macroscopic  Method. 

The  macroscopic  method  can  be  carried  out  with  a  young  living 
culture  on  agar,  or  with  an  emulsion  of  dead  bacilli.  The  technique 
is  not  so  difficult  as  in  the  previous  process,  and  no  apparatus  is 
required  other  than  a  piece  of  narrow  glass  tubing,  from  which  to 
make  pipettes. 

Requisites. — i.  A  young  culture  on  agar,  and  some  normal 
saline  solution  ;  or  a  dead  emulsion  of  typhoid  bacilH. 

2.  Special  glass  pipettes.  These  are  to  be  made  from  a  piece 
of  glass  tubing  which  should  not  be  more  than  ^  inch  in  internal 
diameter  and  about  10  inches  long.  The  central  2  inches  of  this 
tube  are  to  be  heated  in  a  flat  gas  flame  until  thoroughly  softened, 
then  withdrawn,  and  then  the  two  ends  are  to  be  drawn  steadily 
apart.  The  softened  portion  is  to  be  drawn  out  into  a  long,  narrow 
portion  (rather  thicker  than  a  vaccine-tube)  about  6  inches  long 
and  of  nearly  uniform  diameter.  The  full  diameter  of  the  tube 
should  pass  rapidly  into  this  narrow  portion ;  it  should  not  taper 
gradually  (Fig.  14,  c).  The  two  pipettes  thus  obtained  are  to  be 
broken  apart,  leaving  about  3  inches  of  the  narrow  tubing  attached 
to  each.     The  wide  end  of  the  tube  is  to  be  plugged  fairly  firmly 


78  CLINICAL    BACTERIOLOGY    AND    HEMATOLOGY 

with  cotton-wool.  This  serves  a  double  purpose  :  it  prevents  any 
of  the  fluid  from  getting  into  the  mouth,  and  it  offers  a  certain 
amount  of  resistance  to  the  escape  of  the  fluid,  and  thus  renders 
the  pipettes  more  easy  to  fill. 

Process. — Prepare  an  emulsion  just  as  before,  making  a  drachm 
or  so,  and  filtering  it.  An  emulsion  of  dead  bacilli  obtained  from 
a  laboratory  should  be  ready  for  immediate  use.  Insert  the  tip  of 
the  pipette  into  the  blood-serum  (of  which  there  should  be  several 
drops),  and  aspirate  it  gently  into  the  tube,  avoiding  air-bubbles ; 
if  these  gain  access,  blow  out  the  serum  and  begin  again.  It  is 
easier  to  see  what  you  are  doing  if  you  use  a  piece  of  indiarubber 
tubing  (such  as  is  used  with  a  haemocytometer)  through  which  to 
apply  suction. 

Having  drawn  up  enough  serum  to  form  a  column  about  2  inches 
in  length  in  the  narrow  portion  of  the  tube,  lay  the  pipette  on  its 
side,  and  make  a  mark  with  ink  or  with  a  grease  pencil  to  show 
how  high  the  serum  reaches.  Now  suck  the  column  of  fluid  a 
little  way  into  the  tube,  and  insert  the  tip  of  the  pipette  into  the 
emulsion  ;  suck  the  latter  up  the  tube  until  it  reaches  to  the 
mark.  This  will  give  you  the  same  amount  of  emulsion  as  of 
blood-serum ;  the  two  fluids  will  be  separated  by  a  short  column 
of  air.  Now  withdraw  the  tip  for  a  moment  and  suck  up  another 
small  quantity  of  air  ;  dip  it  into  the  emulsion  and  suck  it  up  to 
the  mark  again.  This  will  give  you  twice  the  amount  of  emulsion 
as  of  blood-serum,  the  three  portions  of  fluid  being  separated  by 
air  (Fig.  24).  Repeat  this  process  until  you  have  nine  times  the 
amount  of  emulsion  as  of  serum  ;  then  suck  the  fluid  still  further 
from  the  tip  of  the  pipette,  and  seal  the  latter  in  a  flame. 
Probably  by  this  time  the  fluids  will  have  mixed  together  ;  if  not, 
tap  the  tube  gently  (holding  it  upright)  until  the  air-bubbles 
which  you  have  sucked  up  make  their  escape  and  the  fluid  forms 
a  continuous  column. 

Fill  another  tube  to  a  similar  height  with  emulsion  (for  a 
control),  and  place  the  two  side  by  side  in  an  upright  position  for 
twelve  hours. 

Now  examine  the  fluid  in  the  pipettes.  If  the  emulsion  has 
been  made  from  a  living  culture,  the  control  pipette  {i.e.,  that  to 
which  no  blood  has  been  added)  will  probably  remain  turbid  ;  if 
an  emulsion  of  dead  bacilli  has  been  used,  it  will  have  become 
clear,  and  the  bacilli  will  form  a  uniform,  even  layer  at  the 
bottom  of  the  pipette. 


TYPHOID    FEVER 


79 


^Trdh 


Compare  the  control-tube  with  the  pipette  to  which  blood  has 
been  added.  If  the  reaction  is  negative,  the  appearances  will  be 
exactly  the  same  in  each  ;  but  if  the  reaction  is  positive,  the  bacilli 
will   fall    to   the    bottom    in    flocculent  _^ 

granular  masses,  which  are  similar 
whether  living  or  dead  bacilli  have 
been  used.  In  the  former  case  there 
is  no  difficulty  in  determining  whether 
the  reaction  is  present  or  absent,  for 
the  fluid  will  be  clear  if  it  is  positive, 
turbid  if  it  is  negative  (Fig.  25).  The 
dead  emulsion  presents  more  diffi- 
culties ;  you  have  to  compare  the 
granular  deposit  which  occurs  in  a 
positive  reaction  with  the  more  uniform 
deposit  which  occurs  in  the  control- 
tube.  If  there  is  any  doubt,  the  best 
plan  is  to  break  off  the  tip  of  the  pipette 
and  blow  out  some  of  the  deposit ; 
examine  it  under  the  microscope  after 
applying  a  cover-glass.  In  a  positive 
case  the  bacilli  will  be  seen  to  be  col- 
lected into  clumps ;  in  a  negative  case 
they  will  remain  discrete. 

This  process  is  a  modification  of  that 
described  by  Wright  (Brit.  Med.  Jouvn.^ 
P-  355'  1898),  to  which  the  reader  is 
referred  for  fuller  details. 

It  is  advisable  to  use  a  dilution  of 
I  in  20  if  the  macroscopic  method  is 
used. 

I  now  use  dead  cultures  almost  ex- 
clusively in  routine  work.  They  are 
always  ready,  and  they  do  not  vary  in 
sensitiveness  as  Hving  cultures  do.  The 
method  I  now  use  is  simplicity  itself, 
and  is  specially  suited  for  general 
practitioners,  since  it  requires  abso- 
lutely no  manipulative  skill  and  gives  absolutely  accurate 
results. 

The  method  is,  briefly,  as  follows  :  I  prepare  a  number  of  small 


Fig.  24. — Showing  Method 
OF  FILLING  Pipette. 

a  Is  the  mark  on  the  tube  ; 
b,  the  serum  ;  c,  c,  c,  the 
columns  of  emulsion,  sepa- 
rated by  the  air-bubbles, 
d,  d,  d;  ^  is  the  cotton- 
wool plug. 


8o 


CLINICAL    BACTERIOLOGY   AND    HEMATOLOGY 


sedimentation-tubes  containing  a  definite  bulk  of  killed  typhoid 
emulsion  and  sealed  at  each  end  ;  each  tube  also  contains  a  small 
amount  of  mercury.  Accompanying  each  of  these  tubes  there  is 
a  short  length  of  capillary  tubing  (about  i  inch  long) ;  this  will 
hold  a  definite  fraction  (|,  y^,  gV*  ^tc,  according  to  requirements) 
of  the  amount  of  emulsion  in  the  sealed  tubes.  To  use  them, 
collect  the  blood  in  the  ordinary  way  in  a  Wright's  blood  capsule, 
pipette,  etc.,  and  let  it  coagulate.  It  is  necessary  to  get  clear 
serum  for  the  test,  but  it  is  usually  quite  easy  to  do  this,  even 


Fig.  25. — Positive  and  Negative  Widal's  Reaction  :  Macroscopic 

Method. 

The  fluid  in  the  left-hand  pipette  (negative)  is  still  turbid.  The  right-hand 
pipette  (positive)  contains  clear  fluid,  and  has  a  sediment  at  the  bottom. 
The  lower  portions  of  the  pipette  have  been  removed. 

without  a  centrifuge.  File  and  break  the  pipette  so  that  the 
serum  is  exposed ;  then  take  the  capillary  tube  and  touch  the 
surface  of  the  serum,  which  will  ascend  the  tube  and  just  fill  it. 
File  and  break  off  the  tip  of  the  sedimentation-tube  (at  fe,  Fig.  26), 
drop  in  the  capillary  tube  full  of  serum,  reseal  the  tip  of  the  tube, 
and  allow  it  to  cool.  When  cold,  shake  it  violently  for  half  a 
minute  (when  the  mercury  will  act  as  a  stirrer),  and  put  it  to 
stand  in  a  vertical  position.  The  whole  process  takes  about  two 
minutes,  and  is  about  as  easy  as  testing  for  albumin.  In  positive 
cases  the  first  indication  of  a  reaction  may  be  seen  in  about  an 
hour,  and  it  is  usually  complete  in  four  or  five.     The  fluid  first 


TYPHOID    FEVER 


8l 


shows  a  slight  granularity,  which  becomes  more  and  more  marked, 
minute  greyish  flocculi  separating  themselves  from  the  uniformly 
turbid  emulsion.  These  flocculi  gradually  get  bigger  and  settle 
slowly  at  the  bottom  of  the  tube,  leaving  the  supernatant  fluid 
almost  clear.  The  emulsion  will  settle  by  itself,  but  very  slowly, 
and  falls  as  a  powdery  deposit  quite  different  from  the  flocculi. 
An  unused  sedimentation-tube  (shaken  immediately  before  use)  is 
put  side  by  side  with  the  one  containing  the  serum,  and  used  as  a 
control ;  the  two  are  inspected  from  time  to  time,  and  if  there  is 


Fig.  26. 

a,  Tube  containing  emulsion  of  dead  typhoid  bacilli  and  globule  of  mercury  ; 
b,  showing  method  of  inserting  the  serum  in  a  short  length  of  capillary 
tubing  ;  c,  a  positive  reaction  ;  d,  the  control. 


no  obvious  difference  in  five  or  six  hours  the  reaction  is  absent. 
The  presence  of  the  reaction  in  a  dilution  of  i  in  20  is  fairly 
definite  proof  of  infection. 

The  tubes  are  a  little  trouble  to  prepare,  but  a  stock  sufficient 
to  last  for  months  may  be  made  at  the  same  time.  Prepare 
the  typhoid  emulsion  in  the  method  already  described  (p.  72). 
Next  prepare  some  tubes  of  the  shape  shown  on  Fig.  26, 
sterilize  them  by  dry  heat,  and  place  a  globule  of  mercury  in 
each.     Then  prepare  a  couple  of  Wright's   pipettes   out   of  a 

6 


82  CLINICAL    BACTERIOLOGY   AND    HEMATOLOGY 

piece  of  glass  tubing  about  as  thick  as  a  lead-pencil  and 
about  4  inches  long.  This  is  done  by  holding  the  tube  between 
the  finger  and  thumb  at  each  end  and  softening  it  thoroughly  in  a 
blowpipe  or  Bunsen  flame,  rolling  it  round  and  round  the  while. 
When  the  central  inch  or  more  is  quite  plastic,  the  tube  is  removed 
from  the  flame  and  the  two  ends  drawn  slowly  apart  with  a  steady, 
uniform  pull,  and  held  apart  until  the  glass  is  quite  set.  If  this  is 
quite  successful,  the  softened  portion  should  be  pulled  out  into  a 
tube  about  as  wide  as  a  steel  knitting-needle,  fairly  uniform  in 
diameter,  and  if  possible  2  feet  long  :  this  will  make  two  pipettes 
each  a  foot  long.  To  separate  them,  melt  the  central  portion  in  a 
very  small  flame  (such  as  that  of  a  wax  vesta),  and  pull  them 
apart  rather  quickly.  If  the  capillary  portion  of  your  tube  is 
much  less  than  2  feet  long  it  will  only  serve  for  one  pipette,  and 
the  place  where  it  is  melted  must  be  close  to  one  end.  In  either 
case  the  result  should  be  a  pipette  Hke  that  in  Fig.  36. 

The  most  important  part  of  the  pipette  is  the  tip,  which  is 
represented,  magnified  three  or  four  times,  below  the  pipette.  It 
must  narrow  somewhat  suddenly  into  a  very  fine  capillary  tube  about 
J  inch  long,  so  as  to  oppose  a  considerable  amount  of  friction  to 
the  movement  of  a  column  of  fluid  in  the  tube.  If  the  point  is  a 
good  one,  you  will  have  absolute  control  over  the  column,  and 
will  be  able  to  suck  it  up  to  a  given  point  by  means  of  the  india- 
rubber  nipple  with  absolute  precision.  If  the  capillary  tip  is  too 
narrow  or  too  wide,  draw  it  out  again  until  you  have  one  that  is 
entirely  satisfactory. 

This  is  now  graduated  into  equal  parts  in  the  simple  and 
accurate  method  invented  by  Wright.  Take  a  few  drops  of 
mercury  in  a  short  test-tube,  and  cover  it  with  a  little  water. 
Put  a  mark  with  a  grease  pencil  or  with  pen  and  ink  on  the 
pipette  about  i  inch  from  the  tip :  this  is  for  the  unit.  Then 
take  the  nipple  between  the  thumb  and  finger  of  the  right  hand, 
squeeze  it,  insert  the  tip  in  the  mercury,  and  suck  up  the  latter 
exactly  to  the  mark.  Then  withdraw  the  tip  from  the  mercury 
into  the  water  and  suck  that  up  to  the  mark  ;  the  mercury  will 
rise  in  front  of  it.  When  this  is  done,  replunge  the  tip  into  the 
mercury  and  suck  up  a  second  column.  Repeat  this  process  until 
you  have  filled  the  tube  with  alternate  layers  of  mercury  and  water, 
each  of  exactly  the  same  bulk  (Fig.  27).  When  this  is  done, 
remove  the  tube  from  the  fluid  and  place  it  flat  down  on  the  table, 
still  keeping  suflicient  pressure  on  the  nipple  to  retain  the  fluids 


1 


TYPHOID   FEVER 


83 


in  the  same  place,  so  that  the  junction  of  the  lowest  two  columns 
corresponds  exactly  to  the  unit  mark.  Then  get  a  second  person 
to  make  an  ink  or  grease-pencil  mark  at  the  junction  of  each  of 
the  columns.  Expel  the  fluids,  and  you  will  have  a  Wright^s 
pipette  graduated  into  equal  parts. 

If  you  cannot  suck  up  enough  alternating  layers  to  fill  the  tube, 
suck  up  as  many  as  you  can,  remove  the  pipette,  and  mark  it  as 
before.  Then  relax  the  pressure  so  that  the  whole  column  is 
drawn  upward,  and  adjust  it  until  the  lowest  column  just  coincides 
with  the  uppermost  mark  you  have  just  made,  and  the  rest  of  the 


Fig.  27.— Showing  Method  of  Graduating  a  Wright's  Pipette  in 
Equal  Parts. 


column  extends  into  the  ungraduated  part  of  the  tube  ;  make 
marks  against  the  intersections  as  before. 

Now  proceed  to  put  the  emulsion  into  the  sedimenting-tubes 
with  the  mercury.  If  you  wish  to  prepare  tubes  to  give  you  a 
dilution  of  i  in  20  (and  this  is  the  most  convenient  for  general 
work),  put  as  much  as  will  fill  nineteen  divisions  of  the  pipette, 
sucking  it  up  into  the  pipette  by  using  an  indiarubber  nipple.  If  (as 
is  likely)  your  pipette  does  not  hold  19  units,  it  will  be  necessary 
to  take  two  or  more  pipettes  full — say  10  and  9  units  respectively. 
Then  seal  the  open  end  of  the  sedimenting-tube  in  the  flame,  and 
heat  the  whole  to  60°  C.  for  half  an  hour  to  insure  sterility. 

Lastly,  cut  the  pipette  up  into  lengths  by  notching  it  with  a  file 

6—2 


84  CLINICAL    BACTERIOLOGY   AND    HEMATOLOGY 

opposite  the  marks.  This  will  give  you  a  series  of  short  capillary 
tubes  each  holding  Jy  of  the  volume  of  the  typhoid  emulsion  in 
the  pipettes.  These  are  to  be  used  for  the  serum,  as  already 
described. 

GONORRHCEA 

Nothing  is  more  certain  than  the  fact  that  gonorrhoea  can  only 
be  diagnosed  by  bacteriological  methods  ;  and  every  practitioner 
is  very  strongly  urged  to  practise  himself  in  these  methods  and  to 
employ  them  in  all  cases.  Leaving  out  of  account  the  confidence 
which  the  certainty  of  a  direct  diagnosis  inspires,  there  is  always 
the  possibility  that  legal  questions  may  arise,  and  a  practitioner 
who  made  a  diagnosis  of  gonorrhoea  without  employing  the  only 
means  by  which  that  disease  can  be  diagnosed  would  make  a 
poor  show  in  cross  -  examination.  Lastly,  a  bacteriological 
examination  will  often  tell  us  that  the  disease  is  merely  lying 
latent  and  is  still  infective  when  apparently  cured  ;  but  not  the 
reverse,  for  it  is  not  safe  to  assume  that  the  disease  is  cured 
because  no  gonococci  are  found. 

The  gonococcus  chiefly  aff'ects  mucous  surfaces :  the  urethra 
in  the  male,  the  urethra  and  cervix  uteri  in  the  female,  and  the 
conjunctiva  in  both  sexes.  These  are  the  regions  in  which  the 
primary  lesion  usually  occurs,  and  it  may  extend  by  continuity  to 
more  distant  parts. 

In  the  male  it  may  involve  the  prostate,  the  vesiculae  seminales, 
and  the  bladder.  It  is  doubtful  whether  gonorrhoeal  epididymitis 
is  due  to  this  organism  or  to  another. 

In  the  female  the  inflammation  of  the  urethra  may  extend  to 
the  bladder.  The  inflammation  of  the  cervix  may  extend  to  the 
mucosa  of  the  uterus,  and  thence  to  the  Fallopian  tubes  (causing 
pyosalpinx),  to  the  mouths  of  the  tubes  (causing  local  adhesive 
peritonitis,  which  probably  results  in  sterility),  or  to  the  peri- 
toneum, where  it  may  cause  general  peritonitis. 

The  gonococcus  may  escape  into  the  blood  from  any  of  these 
lesions,  and  the  results  of  this  occurrence  are  arthritis,  ulcerative 
endocarditis,  or  meningitis  ;  the  two  latter  are  rare. 

The  search  for  the  gonococcus  may  have  to  be  made  :  (i)  in 
urethral  pus  from  either  sex  ;  (2)  in  pus  from  the  cervix  uteri ; 
(3)  in  pus  from  the  conjunctiva  ;  (4)  in  pus  from  the  meninges, 
tubes,  peritoneum,  or  other  region,  whether  removed  by  operative 
measures  or  at  a  post-mortem  examination  ;  (5)  in  the  blood ;   or 


GONORRHCEA  85 

(6)  in  the  urine.  It  is  to  be  noticed  that  the  gonococcus  rarely, 
if  ever,  attacks  the  vagina,  except  in  young  children,  and  that  in 
cases  of  vaginitis  the  cervical  secretion  should  be  examined. 

In  the  vast  majority  of  cases  cultural  examinations  are  quite 
unnecessary.  This  is  fortunate,  for  the  gonococcus  does  not  grow 
readily  on  artificial  media.  It  requires  for  its  cultivation  the 
presence  of  haemoglobin,  and  in  practice  the  simplest  method 
(should  cultures  be  required  for  any  purpose)  is  to  smear  sterile 
blood  over  the  surface  of  an  ordinary  agar  tube  and  inoculate  that 
with  the  material  to  be  examined.  To  prepare  these  tubes, 
sterilize  the  tip  of  the  finger  with  carbolic  lotion,  washing  off  the 
latter  with  alcohol  or  ether  ;  then  prick  the  finger  and  squeeze 
two  or  three  drops  of  blood  into  the  tube.  It  will  run  down  the 
medium  and  mix  with  the  water  of  condensation  at  the  bottom. 
Put  the  tube  in  the  incubator  for  twenty-four  hours  to  see  if  it  is 
sterile.  This  will  probably  be  the  case,  as  the  living  leucocytes 
and  fresh  serum  are  probably  sufiicient  to  kill  the  few  stray 
bacteria  that  may  have  entered.  It  is  then  ready  for  use,  and  at 
the  time  of  inoculation  smear  the  blood  over  the  surface  of  the 
agar  with  the  loop.  The  colonies  are  very  small  and  translucent 
(like  those  of  the  pneumococcus),  and  readily  die  out.  The 
organism  has  well-marked  morphological  characters,  and  the 
deductions  drawn  from  these  characters  need  only  be  corroborated 
in  cases  of  generalized  infection  or  of  meningitis,  in  which  the 
results  are  to  be  published  (as  they  should  be),  and  must,  therefore, 
be  proved  beyond  doubt.  In  such  cases  the  services  of  a  bacteri- 
ologist should  be  called  in  if  possible;  or  the  material  may  be 
collected  in  pipettes  with  the  most  careful  precautions  as  to 
asepsis,  and  forwarded  at  once. 

Method  of  Making  the  Films. 

The  pus  is  to  be  spread  out  into  thin  films  at  the  time  at  which 
it  is  taken,  and  this  is  true  whether  the  practitioner  intends  to 
make  the  examination  for  himself  or  is  about  to  send  the  material 
to  a  bacteriologist.  Gonorrhoeal  pus  should  never  be  collected 
on  a  piece  of  cotton-wool  or  enclosed  in  vaccine  tubes. 

The  films  are  to  be  made  thus  :  Take  two  clean  slides  and 
place  two  or  three  platinum  loopfuls  of  the  pus  on  the  centre  of 
one  of  them  ;  sterilize  the  needle  and  lay  it  down.  Now  take  the 
other  slide  and  apply  its  centre  to  the  pus,  and  allow  it  to  fall  on  to 


86  CLINICAL    BACTERIOLOGY    AND    HEMATOLOGY 

the  first  slide  by  its  own  weight ;  do  not  squeeze  the  slides 
together.  Then  slide  them  apart,  keeping  each  in  its  own  plane 
until  they  are  entirely  separated.  This  will  give  you  two  excellent 
films.     Allow  them  to  dry  and  fix  them  in  the  fiame. 

The  films  may  also  be  made  on  cover-glasses,  exactly  the  same 
process  being  adopted,  except  that  it  will  be  necessary  to  squeeze 
the  two  lightly  together.  The  fixation  is  accomplished  by  passing 
the  cover-glasses  rapidly  through  the  flame. 

These  are  the  methods  by  which  films  are  spread  in  all  cases  ; 
the  way  in  which  the  pus  should  be  obtained  varies  somewhat 
with  the  nature  of  the  case. 

In  the  male  it  is  advisable  to  cleanse  the  meatus  and  to  reject 
the  first  drop  of  pus,  taking  the  second  with  a  platinum  loop  and 
proceeding  as  before.  Antiseptic  precautions  are  entirely  un- 
necessary, as  no  attempt  is  to  be  made  to  get  cultures.  If  the 
patient  is  suffering  from  phimosis,  and  there  is  a  purulent 
discharge,  which  may  be  due  to  gonorrhoea,  chancre,  soft  sore,  or 
to  a  non-specific  balanitis,  a  similar  method  is  adopted  ;  but  here 
many  films  should  be  taken,  as  a  prolonged  search  may  be 
required.  If  the  patient  suffers  from  a  slight  discharge  in  the 
early  morning,  the  best  plan  is  to  give  him  two  clean  slides. 
These  are  to  be  smeared  across  the  meatus  whilst  wet  with  the 
discharge,  and  allowed  to  dry. 

In  the  female  it  is  necessary  to  obtain  the  pus  directly  from  the 
urethra ;  it  may  be  expressed  by  the  finger  in  the  vagina.  The 
first  drop  should  be  rejected. 

If  the  patient  is  suffering  from  cervicitis  or  endometritis,  the 
pus  should  be  taken  direct  from  the  cervix,  a  speculum  being 
used,  and  the  pus  being  removed  by  a  platinum  loop  or  probe.  It 
is  necessary  to  emphasize  the  fact  that  the  material  must  be  from 
these  regions  if  a  negative  result  is  to  be  of  any  value.  The  flora 
of  the  vagina  in  all  cases  of  discharge  is  so  extraordinarily 
abundant  that  it  is  almost  impossible  to  recognize  the  gonococcus 
with  certainty  in  such  material. 

It  is  absolutely  necessary  that  you  should  spread  the  films 
at  once,  even  if  you  are  having  the  examination  made  at  a  distance. 
It  is  next  to  useless  to  send  pus  dried  on  linen,  cotton-wool,  a 
Volkmann's  spoon,  or  even  in  a  thick  layer  on  a  slide.  The 
diagnosis  may  be  made  from  material  sent  in  this  way,  but  the 
difficulties  are  much  greater,  and  in  some  cases  the  results  are 
less  certain. 


GONORRHOEA  87 

Preparation  of  films  from  conjunctival  pus  presents  no  diffi- 
culties. The  same  is  true  of  pus  from  the  tubes  or  other  internal 
regions,  whether  it  is  exposed  by  operative  interference  or  at  a 
post-mortem  examination. 

Instructions  for  the  examination  of  the  blood  are  given  subse-_ 
quently.     A  considerable  number  of  films  should  be  taken,  as  the 
cocci  are  present  in  but  very  small  numbers. 

The  urine  may  be  examined  in  the  female  if  a  local  examination 
is  not  considered  advisable,  or  in  the  male  to  obtain  evidences  as 
to  whether  the  disease  is  cured  or  not.  The  morning  urine  should 
be  examined.  It  should  be  mixed  with  a  small  quantity  of  carbolic 
lotion  or  other  antiseptic  and  allowed  to  settle  for  twenty-four 
hours ;  it  is  much  better  to  use  a  centrifuge  if  one  is  available. 
In  cases  where  we  require  evidences  as  to  cure  after  an  attack  of 
gonorrhoea,  the  urine  is  examined  after  gentle  massage  of  the 
prostate. 

Staining  of  Films. 

One  film  is  to  be  stained  by  a  simple  stain  such  as  methylene 
blue  or  carbol  thionin.  The  other  is  to  be  stained  by  Gram's 
method,  and  then  in  dilute  carbol  fuchsin  for  half  a  minute. 

Examination  of  Films. — First  take  the  specimen  in  which  the 
simple  stain  has  been  used,  and  examine  it  with  the  oil-immersion 
lens.  You  will  see  that  it  shows  numberless  cells  with  very 
irregularly  lobed  nuclei ;  these  are  the  pus  cells  or  polymorpho- 
nuclear leucocytes.  There  will  also  be  some  flat  squamous 
epithelial  cells. 

The  gonococci  will  be  stained  even  deeper  than  the  cell  nuclei, 
and  will  be  mostly  contained  within  the  pus  cells.  If  you  see  a 
cell  which  contains  numerous  small  blue  or  violet  granules,  bring 
it  into  the  centre  of  the  field  and  examine  it  more  thoroughly,  to 
see  whether  the  granules  have  the  characters  of  the  organism 
which  we  are  about  to  describe. 

The  gonococcus  is  a  large  diplococcus,  each  component  of  the 
pair  being  shaped  like  a  kidney,  the  hilum  being  turned  toward 
that  of  its  fellow.  Single  forms  (which  may  be  rounded)  and 
tetrads  are  sometimes  seen.  It  does  not  stain  by  Gram's  method, 
and  this  is  one  of  its  most  important  features.  Another  important 
point  is  its  arrangement ;  during  the  height  of  an  attack  of 
gonorrhoea  it  is  almost  entirely  intracellular,  being  contained 
within  the  polymorphonuclear  leucocytes.     Further,  several  pairs 


88  CLINICAL    BACTERIOLOGY    AND    HEMATOLOGY 

occur  in  each  cell,  and  the  great  majority  of  cells  are  entirely 
devoid  of  cocci  (Plate  III.,  Fig.  2). 

If  the  organism  which  you  find  possesses  these  characteristics, 
turn  to  the  specimen  which  has  been  stained  by  Gram's  method 
and  counterstained  by  carbol  fuchsin  (Plate  I.,  Fig.  6).  In  this 
all  bacteria  which  retain  Gram's  stain  will  be  coloured  violet, 
while  organisms  which  do  not  retain  it  will  be  red.  You  must, 
therefore,  search  for  groups  of  diplococci  contained  within  the 
cells  and  possessing  the  above  characteristics.  They  will  not  be 
so  prominent  as  in  the  other  specimen,  for  the  cells,  nuclei,  etc., 
will  be  coloured  red  also,  and  the  contrast  is  not  so  great.  But  if 
the  case  is  one  of  gonorrhoea,  you  will  find  them  after  a  careful 
search. 

If  the  films  came  from  a  female,  you  will  probably  find  the 
appearances  masked  by  numerous  other  organisms,  especially  by 
the  B.  vagina,  a  rather  large  bacillus  (somewhat  resembling 
that  of  anthrax),  which  stains  by  Gram  and  is  often  present  in 
large  numbers,  even  if  the  film  was  taken  in  the  manner  mentioned. 
Yet  in  a  positive  case  you  will  probably  find  the  cells  packed  with 
non-Gram-staining  diplococci  having  the  above  characters  without 
much  difficulty. 

Interpretation  of  Results. 

You  are  justified  in  considering  a  case  to  be  one  of  gonorrhoea 
if  in  films  made  from  the  pus — 

1.  Large  kidney -shaped  diplococci  are  present. 

2.  These  cocci  occur  within  the  pus  cells,  and  at  least  four  pairs 
(Foulerton)  in  each  affected  cell. 

3.  The  vast  majority  of  cells  are  entirely  free  from  cocci.  . 

4.  The  organisms  in  question  do  not  stain  by  Gram's  method. 
This  is  an  absolutely  essential  point. 

If  cocci  are  present  which  answer  to  the  above  description, 
except  that  they  are  not  enclosed  within  cells,  the  case  may  still 
be  one  of  gonorrhoea.  The  gonococcus  is  frequently  extracellular 
during  the  early  stages  of  an  attack  of  urethritis,  and,  though  to  a 
less  extent,  during  its  involution,  whilst  cases  sometimes  occur  in 
which  a  considerable  number  of  the  cocci  lie  free  during  the 
whole  course  of  the  disease. 

In  the  interpretation  of  films  from  a  female,  the  above  criteria 
must  be  insisted  on  very  stringently.  In  the  extraordinary  assort- 
ment of  bacteria  met  with,  if  there  is  an  admixture  of  the  vaginal 


SYPHILIS  8g 

secretion,  you  may  often  find  organisms  resembling  the  gono- 
coccus  in  some  points,  but  not  in  all.  These  yield  one  of  the 
most  troublesome  problems  that  the  clinical  bacteriologist  has 
to  face. 

SYPHILIS 

It  is  now  abundantly  proved  that  the  Spirochata  (or  Treponema) 
pallida  of  Schaudinn  is  the  actual  cause  of  syphilis,  and  the 
diagnosis  of  this  disease  by  the  recognition  of  the  causative  agent 
is  now  practicable  in  some  cases.  The  organism  is  a  spirochaete, 
in  shape  resembling  a  spirillum,  but  probably  of  animal  nature 
{i.e. J  a  protozoon).  It  is  very  small,  or  rather  very  narrow,  and 
stains  with  great  difficulty :  it  is  this  fact  which  has  led  to  its  having 
been  overlooked  previously.  In  length  it  is  about  equal  to  the 
diameter  of  a  red  corpuscle,  either  more  or  less ;  it  is  made  up  of 
about  eight  or  ten  close-set  curves,  and  it  has  sharp  ends.  These 
facts  are  very  important,  for  there  are  numerous  spirochaetes  some- 
what similar  in  appearance,  which  are  frequently  found  in  ulcers 
of  all  sorts,  in  the  mouth,  etc.,  and  which  have  no  doubt  been 
frequently  mistaken  for  Schaudinn's  organism  (Spiroch^ta  pallida). 
The  main  difference  is  that  in  the  other  common  spirochaetes  (one 
of  which,  the  commonest,  is  called  5.  refringens,  and  is  closely 
allied  to,  or  identical  with,  that  of  Vincent)  the  curves  are  wider. 
For  example,  if  we  found  two  spirochaetes  exactly  as  long  as  a 
red  corpuscle  is  wide,  and  in  one  there  were  eight  complete  curves 
and  in  the  latter  only  three  or  four,  the  former  would  probably  be 
pallida,  the  latter  refringens  (Plate  V.).  The  former  is  also  said 
to  look  stiffer  and  to  be  less  easily  bent.  The  staining  reactions  are 
different  in  the  two  cases,  refringens  being  stained,  though  not 
deeply,  with  borax-methylene  blue  or  dilute  carbol  fuchsin,  whilst 
the  latter  is  not.  ' 

Method. — The  examination  may  be  carried  out  on  scrapings 
from  a  supposed  chancre,  secondary  ulcer,  condyloma,  etc. ;  on 
juice  obtained  from  an  enlarged  lymphatic  gland  by  puncture  with 
a  hypodermic  needle ;  on  the  blood  expressed  from  a  secondary 
rash  after  puncture  of  the  skin,  or,  according  to  some  authors,  in 
fluid  from  a  blister  raised  on  or  near  a  lesion  of  such  a  rash.  It 
appears  to  be  especially  abundant  in  the  pemphigoid  rashes  of 
hereditary  syphilis.  It  is  very  rarely  found  in  gummata  or  in 
any  tertiary  lesion. 

Spread  the  material  thus  obtained  in  a  very  thin  layer  on  a 


go  CLINICAL    BACTERIOLOGY    AND    HEMATOLOGY 

scrupulously  clean  slide,  allow  to  dry,  and  fix  by  gentle  heat — 
i.e.f  such  that  it  does  not  get  uncomfortably  hot  to  the  finger. 

Prepare  a  mixture  of  lo  c.c.  tap- water  (or  of  distilled  water 
+  I  drop  of  a  I  in  i,ooo  solution  of  potassium  carbonate),  and 
lo  drops  of  Giemsa's  stain,  w^hich  must  be  bought  ready  pre- 
pared. In  mixing  the  two  avoid  violent  agitation ;  add  the  stain 
to  the  water  in  a  test-tube,  stopper  the  latter  with  your  thumb  and 
invert  it  slowly  once  or  twice.  Do  not  mix  it  until  you  are  ready 
to  proceed  with  the  staining. 

Take  the  slide  in  a  perfectly  clean  pair  of  forceps,  flood  it  with 
the  stain,  and  heat  until  it  just  begins  to  steam  ;  remove  it  from 
the  flame  and  in  fifteen  seconds  pour  off"  the  solution,  replace  it 
quickly  with  fresh  and  heat  again,  again  removing  it  when  steam 
rises  and  allowing  the  action  to  go  on  for  fifteen  seconds.  Do 
this  four  times  in  all,  allowing  the  action  to  go  on  for  one  minute 
on  the  last  application.  Then  wash  in  tap- water  or  in  distilled 
water  with  a  drop  or  two  of  potassium  carbonate  solution,  blot, 
dry,  and  mount. 

Another  method  is  to  place  the  slide  face  downwards  in  a  Petri 
dish,  supported  on  two  slips  of  glass.  The  dish  is  then  filled  with 
a  mixture  prepared  as  above,  and  the  staining  allowed  to  go  on 
for  twelve  to  twenty  hours  in  the  cold,  or  three  to  four  hours  in 
the  incubator,  the  dish  being  covered  to  prevent  evaporation.  If 
the  slide  is  inserted  face  upwards,  it  will  probably  be  covered  by 
a  fine  red  precipitate. 

The  most  minute  amount  of  acid  is  fatal  to  the  process ;  hence 
all  instruments  must  be  dry  and  clean,  and  distilled  water  (which 
often  contains  traces  of  acids)  should  be  avoided. 

Examine  the  film  with  the  ^^  and  your  highest  eyepiece,  taking 
great  care  to  get  a  good  light,  white  if  possible. 

Another  method,  with  which  I  have  been  fairly  successful,  and 
which  is  strongly  recommended  by  Herxheimer,  consists  in 
fixing  as  before,  and  allowing  the  film  to  stain  for  twenty-four 
hours  in  a  I  in  i,ooo  solution  of  Nile  blue  in  distilled  water.  The 
spirochaetes  are  stained  a  fine  blue,  and  are  readily  recognizable. 

In  making  the  search  a  good  lens  and  good  light  are  always, 
and  much  patience  frequently,  necessary ;  the  spirochaetes  may 
be  but  one  or  two  on  a  film,  or  there  may  be  several  on  one  field 
of  the  microscope.  Very  occasionally  they  are  matted  together 
in  a  dense  mass.  Having  found  a  spirochaete,  proceed  to  see  if 
it  resembles  the  pallida  or  the  refringens  ;   note  especially  its 


CHOLERA  91 

ength  (comparing  it  with  a  red  blood-corpuscle)  and  the[number  of 
turns  of  which  this  distance  is  made  up.  If  there  are  about  six 
or  eight  turns  to  this  distance,  it  is  almost  certainly  pallida ;  if 
there  are  fewer,  it  is  not.  Examine  its  ends,  and  see  whether  they 
taper  off  to  a  point  or  terminate  abruptly. 

It  is  also  possible  to  see  the  spirochaete  unstained,  when  it  is 
actively  motile,  but  it  is  not  possible  to  distinguish  it  from  its 
congeners  in  this  way  without  much  practice.  In  point  of  fact 
practice  is  an  essential  in  the  diagnosis  of  syphilis  by  the  recognition 
of  the  spirochaete,  and  the  practitioner  is  recommended  to  identify 
the  organism  as  often  as  he  can  in  undoubted  cases  before 
attempting  its  recognition  in  doubtful  ones  for  diagnostic  purposes. 

CHOLERA 

The  diagnosis  of  cholera  can  only  be  made  on  clinical  grounds 
alone  during  an  epidemic,  as  other  diseases  present  almost 
identical  symptoms  and  course.  The  importance  of  making  a 
correct  diagnosis  arises  less  from  the  interests  of  the  patient  than 
from  those  of  the  general  public  ;  if  the  case  is  one  of  true  Asiatic 
cholera,  the  sanitary  authorities  must  be  notified  and  the  fullest 
precautions  taken  to  prevent  the  spread  of  the  disease.  In  all 
suspicious  cases  a  quantity  of  the  rice-water  stools  (in  a  bottle 
sterilized  by  boiling  or  by  dry  heat  and  securely  packed)  should  be 
forwarded  at  once  to  a  public  laboratory.  Meanwhile  the 
diagnosis  may  be  established  with  a  fair  amount  of  certainty  by 
the  following  simple  tests : 

1.  Take  a  platinum  loopful  of  the  dejecta  and  spread  it  in  a 
thin  film  on  a  clean  slide ;  dry,  fix,  and  stain  with  carbol  fuchsin 
for  three  minutes  ;  wash,  dry,  and  mount. 

2.  Prepare  another  film  and  stain  by  Gram's  method. 

Examination  of  the  Films. 

The  spirillum  of  Asiatic  cholera  is  about  half  as  long  as  a 
tubercle  bacillus,  or  rather  longer,  and  much  thicker.  It  is 
slightly  curved  :  hence  the  name  of  the  "  comma  bacillus."  It 
looks  very  like  a  caraway  seed  (Plate  II.,  Fig.  5). 

In  the  carbol  fuchsin  specimen  vast  numbers  of  these  curved 
rods  will  be  seen ;  probably  few  other  organisms,  if  any,  will  be 
present  if  the  case  is  one  of  true  cholera.  Two  or  more  rods 
may  often  be  found  joined  together  with  their  concavities  turned 


92  CLINICAL    BACTERIOLOGY   AND    HEMATOLOGY 

in  Opposite  directions,  giving  the  whole  the  appearance  of  a 
very  elongated  spiral.  In  the  stools  (but  not  usually  in  cultures) 
the  individual  rods  have  frequently  a  parallel  arrangement,  pre- 
senting the  appearance  of  "  shoals  of  fish  swimming  up  stream." 

If  you  see  these  appearances  examine  the  Gram  specimen. 
Very  few  organisms  will  be  visible,  as  the  cholera  vibrio  does 
not  retain  the  stain  when  treated  in  this  way. 

If  vibrios  having  the  above  characters  are  present,  proceed  as 
follows : 

Take  two  or  three  small  flasks  (preferably  sterilized  by  heat), 
and  add  to  each  loo  c.c.  of  water,  i  gramme  of  peptone,  and 
J  gramme  of  common  salt ;  boil  thoroughly,  and  allow  to  cool. 
This  forms  a  culture  medium  in  which  the  cholera  vibrio  will 
grow  very  rapidly,  and  other  organisms  far  more  slowly. 

Inoculate  each  flask  with  a  loopful  or  two  of  dejecta,  plug 
each  with  cotton-wool,  and  incubate  for  eight  to  twelve  hours  at 
37°  C.  If  cholera  vibrios  are  present  the  cultures  will  conform  to 
the  following  tests  : 

(a)  There  will  be  a  film  on  the  surface.  This  will  be  more 
marked  after  a  few  hours  longer. 

{b)  This  scum  will  present  the  microscopic  appearances 
described  above,  except  that  the  vibrios  are  usually  somewhat 
straighter  than  those  which  occur  in  the  stools,  and  the  "  fish-in- 
stream"  arrangement  is  not  marked.  They  will  not  stain  by 
Gram's  method. 

(c)  The  addition  of  a  small  quantity  of  pure  strong  sulphuric 
acid  will  give  a  pink  or  crimson  tint.  This  is  the  "  cholera-red  " 
reaction,  and  is  caused  by  the  action  of  sulphuric  acid  on  indol 
in  the  presence  of  a  minute  quantity  of  a  nitrite;  many  other 
organisms  {e.g.,  the  B.  coli)  produce  this  colour  after  the  addition 
of  a  nitrite,  very  few  without  it.  The  cholera  vibrio  produces 
nitrites  as  well  as  indol. 

Interpretation  of  Results. 

In  a  case  in  which  the  above  phenomena  are  observed,  the 
inference  that  the  patient  is  suffering  from  true  Asiatic  cholera  is 
so  strong  that  the  authorities  should  be  notified  and  the  fullest 
precautions  taken. 

A  case  in  which  they  are  absent  is  almost  certainly  not  one  of 
true  cholera. 


PLAGUE  93 


PLAGUE 


The  bacteriological  diagnosis  of  plague  should  be  made  by  an 
expert ;  not  because  it  is  difficult,  but  because  so  much  hinges 
upon  it — at  least,  in  this  country.  A  brief  account  of  the  method 
by  which  a  practitioner  who  was  unable  to  obtain  expert  help 
might  proceed  may  not  be  out  of  place. 

The  plague  bacillus  is  a  short  and  rather  thick  rod  which  occurs 
in  vast  numbers  in  the  bubo,  in  the  blood,  and  in  the  internal 
organs.  It  does  not  stain  by  Gram's  method,  and  when  stained 
by  other  processes  it  often  exhibits  a  characteristic  polar  staining, 
the  ends  of  the  bacillus  being  coloured  deeply,  whilst  the  inter- 
vening portion  remains  colourless  (Plate  II.,  Fig.  4).  It  might 
be  mistaken  for  a  diplococcus ;  it  could  not  be  mistaken  for 
the  pneumococcus  (to  which  it  has  some  resemblance),  as  that 
organism  stains  by  Gram.  Degenerate  forms  which  resemble 
cocci,  etc.,  often  occur  in  cultures,  but  are  seldom  met  with  in 
the  body  during  life. 

The  diagnosis  may  be  made  by  an  examination  of  fluid 
aspirated  from  the  bubo  or  of  the  blood.  In  bubonic  cases  the 
former  method  should  always  be  adopted,  as  the  bacilli  are 
present  therein  in  vast  numbers,  and  generally  in  pure  culture  ; 
the  amount  of  fluid  which  has  to  be  removed  is  very  small,  even 
if  cultures  have  to  be  taken. 

When  this  is  not  the  case  two  films  should  be  made  in  the  way 
already  described,  fixed  and  stained,  the  one  by  dilute  carbol 
fuchsin  or  Loffler's  blue,  and  the  other  by  Gram's  method.  If 
the  bacilli  are  present,  they  will  appear  as  short  oval  rods  which 
may  or  may  not  exhibit  the  polar  staining  ;  if  the  specimen  has 
been  stained  for  the  proper  length  of  time  (about  two  minutes), 
most  of  them  will  do  so,  but  in  any  case  it  will  most  likely  be 
present  in  a  few.  The  Gram  specimen  will  not  show  these  rods  ; 
there  may  be  a  few  pus  cocci  present  as  a  secondary  infection. 

The  blood  is  examined  by  any  of  the  methods  to  be  described 
subsequently,  and  a  very  careful  search  made,  as  the  numbers  of 
the  bacilli  may  be  comparatively  scanty. 

If  a  careful  examination  of  stained  films  made  from  a  bubo 
does  not  show  the  organisms  having  the  above  characters,  it  is 
probably  safe  to  say  that  the  case  is  not  one  of  plague. 


94  CLINICAL    BACTERIOLOGY    AND    HEMATOLOGY 


SOFT  SORE 

A  very  strong  case  has  been  made  out  in  favour  of  the  view 
that  the  bacillus  described  by  Unna  and  by  Ducrey  is  actually 
the  cause  of  soft  sore,  though  the  proof  does  not  rest  upon  the 
solid  ground  demanded  by  Koch.  The  bacillus  has  never  been 
cultivated  outside  the  body,  and  the  chief  proof  of  its  pathogenicity 
rests  upon  the  fact  of  its  invariable  presence  in  true  soft  sores. 
It  is  hardly  correct  to  say  that  it  is  invariably  absent  from  other 
sores,  unless,  indeed,  we  extend  somewhat  our  conception  of  what 
a  soft  sore  really  is  ;  for  the  author  has  found  bacilli  which  were 
indistinguishable  from  these  in  sores  which  were  exactly  like 
those  which  follow  herpes  preputialis,  though  more  severe  than 
is  generally  the  case.  But  it  is  quite  possible  that  future  research 
will  show  that  the  bacillus  varies  greatly  in  virulence,  and  that  it 
is  only  under  certain  circumstances  that  it  produces  typical  soft 
sores.  At  present  all  we  can  say  about  the  diagnostic  value  of 
the  organism  is  that  its  presence  in  the  secretion  from  an  ulcer 
affords  strong  evidence  that  the  case  is  really  one  of  chancroid, 
and  that  its  ab.ence  almost  certainly  nullifies  such  a  diagnosis. 
It  is  scarcely  necessary  to  say  that  syphilis  and  soft  sore  (or 
gonorrhoea  and  soft  sore)  may  be  inoculated  at  the  same  time, 
and  the  lesions  appropriate  to  both  diseases  may  be  present 
simultaneously. 

The  bacillus  in  question  is  a  short  straight  rod,  less  than  a 
quarter  the  length  of  the  tubercle  bacillus,  and  not  much  longer 
than  the  bacillus  of  influenza.  It  is  rather  thick  in  proportion  to 
its  length,  its  length  being  only  about  three  times  its  breadth,  and 
it  has  rounded  ends,  which  often  stain  more  deeply  than  the 
centre  of  the  bacillus.  It  does  not  stain  by  Gram's  method ;  it 
stains,  indeed,  with  some  difficulty,  and  powerful  stains  (such  as 
dilute  carbol  fuchsin  or  Loffier's  blue)  should  be  used.  It  is  best 
demonstrated  in  films  made  from  the  deeper  parts  of  a  typical 
soft  sore,  for  the  superficial  parts  contain  bacteria  of  all  sorts, 
and  the  recognition  of  Unna's  bacillus  is  not  easy  unless  it  is 
obtained  in  large  numbers. 

The  bacteriological  examination  for  this  bacillus  has  most  often 
to  be  made  in  cases  of  urethral  sore,  or  of  a  sore  concealed 
beneath  a  phimosis.  The  method  of  obtaining  the  specimen  is 
the  same  in  both  cases.     A  fairly  stiff  platinum  loop  is  inserted 


SOFT    SORE — RINGWORM  95 

beneath  the  prepuce,  or  into  the  urethra,  and  moved  gently  about 
until  the  most  tender  spot  is  found.  This  should  be  scraped  as 
forcibly  as  the  patient  will  allow,  and  the  loop  withdrawn,  care 
being  taken  that  the  mass  of  secretion  is  not  wiped  off  in  so  doing. 
Several  films  should  then  be  made,  the  secretion  being  rubbed  up 
on  the  slide  with  a  drop  of  water.  They  should  be  stained  with 
either  of  the  stains  mentioned  above  for  five  minutes  or  more  : 
it  is  an  advantage  to  warm  them  gently.  They  are  then  rinsed 
in  water,  dried  and  mounted,  and  examined  thoroughly  with  an 
oil-immersion  lens. 

When  buboes  occur  in  the  course  of  soft  sore,  the  pus  they 
contain  should  be  examined  for  this  organism  as  soon  as  they  are 
opened  ;  the  interest  in  this  is  chiefly  scientific,  for  opinions  are 
divided  as  to  whether  they  are  caused  by  this  germ  or  by  pyogenic 
bacteria.  In  three  cases  examined  by  the  author  it  was  found  (in 
very  scanty  numbers)  on  one  occasion,  staphylococci  on  another, 
and  no  bacteria  of  any  sort  in  the  third. 

RINGWORM 

An  examination  of  the  hair  and  scales  from  the  skin  is  of  very 
great  value  to  the  dermatologist.  It  settles  conclusively  the 
question  whether  a  patient  is  or  is  not  suffering  from  ringworm, 
and  often  gives  important  information  as  to  prognosis,  and  may 
enable  us  to  say  whether  the  disease  has  probably  been  derived 
from  a  human  source,  or  has  been  contracted  from  one  of  the 
lower  animals. 

Where  the  mere  diagnosis  of  ringworm  is  in  question,  an 
examination  of  the  hair  or  scales  after  soaking  in  liquor  potassae 
is  often  sufficient.  The  materials  are  placed  in  a  drop  of  the 
solution  on  a  slide,  and  covered  with  a  cover-glass  ;  after  a  quarter 
of  an  hour  or  so  the  specimen  is  carefully  examined  under  the 
microscope,  using  a  J-inch  lens  and  a  small  diaphragm.  The 
spores  appear  as  spherical  or  oval,  highly  refractile  bodies,  which 
can  hardly  be  mistaken  for  anything  but  fat  globules.  This 
possible  source  of  fallacy  may  be  removed  by  soaking  the  hair 
in  ether  before  applying  the  liquor  potassae. 

This  is  a  rapid  and  simple  method,  but  it  does  not  enable  us  to 
diagnose  the  nature  of  the  fungus  with  certainty,  except  in  very 
typical  cases,  and  when  spores  are  scanty  they  are  readily  over- 
looked.    It  is  a  very  great  advantage  to  employ  some  method  of 


96  CLINICAL    BACTERIOLOGY   AND    HEMATOLOGY 

Staining.  These  are  by  no  means  difficult,  though  they  are  some- 
what tedious.  Two  processes,  both  modifications  of  Gram's  method, 
will  be  given.     They  are  not  difficult  to  apply,  but  are  very  slow. 


Method  of  Staining  Hairs,  Scales,  etc 

Requisites. — i.  Aniline  gentian  violet^  carbol  gentian  violet,  or 
carbol  fuchsin. 

2.  Gram's  iodine  solution. 

3.  Aniline  oil,  to  which  a  little  strong  hydrochloric  acid  has  been 
added,  two  or  three  drops  to  a  quarter  of  a  test-tube  full  of  the  oil. 
Prepare  when  required,  and  shake. 

4.  Aniline  oil. 

5.  Xylol.  This  is  not  necessary  unless  the  specimens  are  to 
be  kept  permanently. 

6.  Blotting-paper. 

7.  Slides,  cover-glasses,  and  balsam. 

Process. — Take  a  broken  hair  or  stump  from  the  edge  of  the 
suspected  area,  and  cut  off  all  the  free  portion,  except  a  piece 
about  \  inch  long.  The  root  and  the  part  of  the  shaft  next 
to  it  are  all  that  are  to  be  examined.  Several  of  these  hairs  can 
be  stained  at  the  same  time. 

Stain  in  a  watch-glass  full  of  stain,  or  on  a  slide,  for  a  quarter 
of  an  hour,  or  longer — the  longer  the  better ;  it  is  an  advantage  to 
warm  the  stain  gently,  and  it  will  penetrate  better  if  the  hairs 
have  been  previously  washed  in  ether,  though  this  is  not  absolutely 
necessary. 

Remove  the  hairs  from  the  stain,  and  place  them  on  a  slide ;  if 
already  on  a  slide,  pour  off  the  stain.  Dry  them  with  blotting- 
paper,  and  pour  on  the  iodine  solution ;  allow  it  to  act  for  five 
or  ten  minutes.  It  is  an  advantage  to  blot  the  specimen  after  a 
minute  or  two,  and  apply  a  fresh  lot  of  the  solution.  Blot  thoroughly. 

Now  decolorize  in  the  solution  of  hydrochlorate  of  aniline  in 
aniline  oil.  This  should  be  poured  off  and  replaced  occasionally, 
and  the  specimen  examined  under  a  low  power  of  the  microscope. 
When  the  decolorization  appears  to  be  complete  (it  may  take 
many  hours),  pour  off  the  solution  and  replace  it  by  aniline  oil ; 
allow  this  to  act  for  an  hour  or  more,  renewing  it  occasionally. 

If  the  specimen  is  not  to  be  kept  permanently  it  may  now  be 
mounted  in  balsam  and  examined  at  once.     If  it  is  to  be  kept  the 


RINGWORM  97 

aniline  oil  must  be  washed  out  by  several  applications  of  xylol. 
Mount  in  balsam. 

Adamson's  method  gives  good  results,  but  is  somewhat  more 
tedious.  _ 

Requisites. — i.  Liquor  potassae. 

2.  Dilute  alcohol — about  15  per  cent. 

3.  Aniline  gentian  violet  or  its  substitutes. 

4.  Gram's  iodine  solution. 

5.  Aniline  oil. 

6.  Xylol. 

7.  Blotting-paper. 

8.  Slides,  cover-glasses,  and  balsam. 

Process. — Prepare  the  fragments  of  hair  as  before,  rejecting  the 
free  portions.  Place  them  on  a  slide,  add  a  drop  or  two  of  liquor 
potassae,  and  apply  a  cover-glass.  Allow  the  liquor  to  act  for  a 
quarter  of  an  hour  or  twenty  minutes. 

Now  place  a  large  drop  of  dilute  spirit  at  one  edge  of  the  cover- 
glass,  and  a  piece  of  blotting-paper  at  the  opposite  edge  ;  this 
will  suck  up  the  potash,  and  the  spirit  will  run  in  and  replace  it. 
After  a  few  minutes  lift  over  the  cover-glass,  and  wash  the  hairs 
gently  in  more  spirit.  This  will  harden  them.  Dry.  If  epithelial 
scales  are  being  examined,  they  may  be  fixed  to  the  slide  or  cover- 
glass  by  heat  in  the  usual  way. 

Stain  in  aniline  gentian  violet  for  half  an  hour  or  less. 
■  Pour  off  the  stain,  blot  gently,  and  pour  on    Gram's  iodine 
solution.     Allow  this  to  act  for  five  minutes.     Blot  again. 

Decolorize  with  aniline  oil,  pouring  it  off  and  applying  a  fresh 
lot  from  time  to  time.  The  process  may  take  an  hour  or  more, 
and  the  specimen  should  be  left  under  the  microscope  and  ex- 
amined occasionally. 

When  the  decolorization  is  complete  {i.e.^  when  the  colour  is 
seen  to  be  present  in  the  fungus  only),  blot  gently,  and  wash 
thoroughly  with  xylol.     Mount  in  balsam. 

The  specimens  are  to  be  examined  under  a  ^-inch  objective. 
A  higher  power  is  unnecessary. 

There  are  certainly  three,  and  possibly  more,  species  of  ring- 
worm which  occur  in  England,  and  the  fungus  of  favus  is  closely 
allied,  and  is  demonstrated  by  the  same  process. 

The  MicrospoYon  Audouini  is  the  most  common  species  of  ring- 
worm fungus  in  this  country,  being  responsible  for  about  80  or 
90  per  cent,  of  all  cases.     It  is   the  small-spored  fungus,  and 

7 


98  CLINICAL    BACTERIOLOGY   AND    HEMATOLOGY 

it  may  be  distinguished  by  the  fact  that  its  spores  are  arranged 
in  an  irregular  mosaic,  and  not  in  chains.  Its  mycelium,  which 
consists  of  long  threads,  lies  in  the  interior  of  the  hair,  whilst 
the  spores  form  a  thick  mass  outside  (Fig.  28,  and  Plate  VI., 
Fig.  4).  This  sheath  of  spores  projects  a  short  distance  above 
the  surface,  and  may  often  be  seen  with  the  naked  eye.  The 
outside  of  the  hair  is  destroyed,  and  the  surface  of  the  hair 
eroded ;  the  former  feature  serves  to  differentiate  it  from  all 
other  varieties  of  ringworm,  and  from  favus. 

This  fungus  usually  attacks  the  scalp  in  children,  and  in  them 
it  dies  out  spontaneously  about  the  age  of  sixteen,  whilst  other 


Fig.  28. — MiCROSPORON  Audouini.* 

varieties  of  ringworm  often  do  not.  It  is  very  rare  in  adults,  and 
it  rarely  attacks  other  regions  than  the  head.  It  is  thought  by 
some  to  be  always  caught  from  a  human  case  of  the  disease,  but 
there  are  reasons  for  thinking  that  it  may  be  derived  by  infection 
from  the  horse  or  cat. 

The  most  important  clinical  fact  about  the  M.  Audouini  is  that 
ringworm  caused  by  it  is  extremely  intractable,  and  may  run  a 
prolonged  course  in  spite  of  the  most  skilful  treatment. 

The  Trichophyton  (or  Megalosporon)  endothrix  attacks  the  interior 
of  the  hairs,  and  forms  long  chains  ;  it  always  involves  the  hair  just 
inside  the  cuticle,  being  in  reality  an  endo-ectothrix.  Its  spores 
are  somewhat  larger  than  those  of  the  preceding  species,  but  the 
difference  is  not  great ;  the  organism  is  most  easily  recognised  by 
the  chain-like  arrangement  of  its  spores,  and  by  the  fact  that 

*  Fig.  28  is  from  Curtis's  "  Essentials  of  Bacteriology  "  (Longmans), 


RINGWORM  99 

they  lie  within  the  hair,  the  cuticle  of  which  usually  remains 
intact  (Plate  VI.,  Fig.  2). 

This  fungus  is  a  rare  cause  (in  this  country)  of  ringworm  of  the 
scalp,  and  the  disease  caused  by  it  appears  to  be  somewhat  easier 
to  cure  than  that  due  to  the  microsporon,  though  opinions  on  this 
point  are  divided.  It  also  attacks  other  regions  of  the  body, 
causing  tinea  circinata,  or  eczema  marginatum. 

It  always  appears  to  be  derived  from  a  human  case,  and  never 
by  infection  from  animals. 

The  T.  ectothfix  (like  the  microsporon)  forms  a  sheath  round 
the  outside  of  the  hair,  to  which  it  is  closely  applied,  like  the  bark 
to  a  tree,  but,  unlike  the  small-spored  variety,  does  not  destroy 
the  cuticle.  The  spores  are  about  as  large  as  in  the  preceding 
species,  and  are  arranged  in  chains ;  this  fact,  together  with  the 
position  of  the  fungus  with  regard  to  the  hair,  will  enable  a 
diagnosis  to  be  made  (Plate  VI.,  Fig.  i). 

This  fungus  is  a  rare  cause  of  ringworm  of  the  scalp,  and  the 
disease  it  causes  is  readily  cured.  It  also  causes  ringworm  of 
the  body  and  of  the  beard  region,  which  the  endothrix  does  not 
attack.  According  to  Sabouraud  (to  whose  work  on  these 
organisms  we  owe  most  of  our  knowledge  on  the  subject),  kerion 
is  always  caused  by  this  organism,  but  this  is  not  generally 
accepted.  It  appears,  however,  to  be  a  fact  that  suppurative 
lesions  (folliculitis,  kerion,  etc.)  are  usually  caused  by  this  fungus. 

This  species  is  often  derived  from  one  of  the  lower  animals, 
especially  from  the  horse,  cat,  and  dog. 

Favus  is  caused  by  a  closely  allied  organism,  the  Achovion 
Schonleinii.  This  may  be  demonstrated  by  either  of  the  processes 
already  described.  It  affects  the  skin  in  two  ways  :  by  the  forma- 
tion of  the  characteristic  scutula  and  by  the  ringworm-like  invasion 
of  the  hair.  The  scutulum  is  composed  of  vertical  mycelial  fila- 
ments, which  branch,  and  which  appear  to  be  composed  of  short 
rods.  There  are  often  oval  spores  at  the  free  ends  of  these 
branches,  and,  according  to  Sabouraud,  branching  into  three  occurs 
(trichotomous  branching),  and  is  very  characteristic,  though  diffi- 
cult to  see.  The  radiation  of  several  filaments  from  one  point, 
and  the  fact  that  these  appear  to  be  made  up  of  short  lengths, 
gives  rise  to  an  appearance  which  has  been  compared  to  that  of 
the  metatarsal  bones,  and  the  term  "  favic  tarsus"  has  been 
appUed. 

When  favus  affects  the  hair  (Plate  VI.,  Fig.  5),  the  cuticle 

7—2 


TOO  CLINICAL    BACTERIOLOGY    ANG    HEMATOLOGY 

remains  intact,  and  the  inner  portion  of  the  hair  is  packed  with 
long  waving  filaments,  whilst  the  outside  (under  the  cuticle)  is 
covered  with  short  "elements" — the  exact  nature  of  which  is 
doubtful — of  every  shape  and  size. 

The  identification  of  the  variety  of  organism  which  is  present 
is  not  usually  difficult.  The  first  point  to  be  looked  to  is  the 
arrangement  of  the  spores.  If  these  form  an  irregular  mosaic, 
the  microsporon  is  present ;  if  they  form  filaments  somewhat 
resembling  those  of  a  streptococcus,  the  organism  is  a  tricho- 
phyton. Then  look  to  see  whether  the  cuticle  is  intact,  and 
whether  the  fungus  invades  the  interior  of  the  hair.  This 
can  usually  be  determined  by  focussing  up  and  down  until 
you  see  an  *'  optical  section  "  of  the  hair  in  question.  It  is 
important  not  to  be  deluded  by  the  fact  that  the  fungus  which 
lies  on  the  outside  of  the  hair  will  appear  to  be  inside  it  if  a 
surface  view  only  is  taken. 

Ringworm  of  the  Nails. 

The  nails  may  be  affected  by  either  of  the  trichophytons  or  by 
favus.  The  diagnosis  of  the  presence  of  ringworm  may  usually 
be  made  by  the  examination  of  pieces  of  the  nails  after  soaking  in 
liuqor  potassae,  but  a  prolonged  examination  of  many  pieces  must 
be  made  before  their  absence  is  assumed.  The  diagnosis  of  the 
variety  present  can  only  be  made  by  cultures,  and  is  not  of  cHnical 
importance  (Plate  VI.,  Fig.  3). 


OTHER  SKIN  DISEASES 

In  seborrhoeic  affections  the  bottle  bacillus  of  Unna  is  constantly 
present,  and  as  it  does  not  occur  at  ail  frequently  in  other  diseases, 
if  at  all,  it  is  a  useful  test  between  seborrhoeic  dermatitis  and 
psoriasis,  especially  on  the  scalp.  It  is  a  moderately  large 
organism  which  consists  of  an  oval,  spore-like  body  attached  to  a 
short  neck  or  handle.  The  former  stains  faintly,  especially  in  the 
middle,  where  it  seems  to  have  a  clear  vacuole,  whilst  the  handle 
stains  deeply.  It  is  very  easily  recognised  after  it  has  once  been 
seen ;  it  is  perhaps  the  only  bacterium  which  can  be  positively 
identified  by  microscopic  means  alone.     It  stains  by  Gram. 

To  search  for  it,  take  a  scale  or  two  from  the  affected  region  and 
grind  it  in  a  drop  of  water  between  two  slides  until  reduced  to  a 


OTHER  SKIN   DISEASES  TOI 

pulp.  Allow  some  of  this  pulp  to  dry  on  one  of  the  slides,  fix, 
stain  by  Gram's  method,  and  do  not  counterstain.  If  the  film  is 
very  greasy,  so  that  the  stain  does  not  wet  it,  warm  the  slide 
gently  and  allow  a  few  drops  of  ether  to  flow  over  the  film,  fix^ 
again,  and  proceed  as  before.  Examine  the  preparation  under 
a  j-^^-inch.  The  presence  of  the  bottle  bacillus  is  almost  conclusive 
evidence  in  favour  of  seborrhoea,  as  against  psoriasis,  syphilis,  etc. 

The  figure  (Plate  IV.,  Fig.  6)  I  owe  to  the  kindness  of 
Dr.  Whitfield.  It  is  from  an  impure  culture,  the  only  one  that 
has  ever  been  obtained. 

Tinea  Versicolor. — There  is  usually  no  difficulty  in  the  diag- 
nosis of  this  disease  by  ordinary  clinical  methods.  Where  there  is 
any  doubt,  one  of  the  scales  may  be  removed  and  examined  in  liquor 
potassae,  or  by  any  of  the  methods  described  for  ringworm. 
The  fungus — M.  furfur — is  readily  detected  under  a  |^-inch.  It 
consists  of  rather  wide  mycelial  threads,  branching  and  inter- 
lacing, with  masses  of  refractile  spore,  looking  like  bunches  of 
grapes  (Plate  IV.,  Fig.  4). 


SECTION  III 

COLLECTION  AND  EXAMINATION  OF  CERTAIN 
MORBID  MATERIALS 

THE  COLLECTION  AND  EXAMINATION  OF  PUS 

When  a  simple  microscopical  examination  has  to  be  made,  the 
collection  of  pus  presents  no  difficulties,  as  the  few  bacteria 
which  may  gain  access  from  the  skin  or  the  air  will  not  lead  to 
error.  The  case  is  otherwise  where  cultural  examinations  have 
to  be  made,  or  where  the  material  has  to  be  taken  to  a  laboratory. 
Here  the  material  should  be  collected  in  a  pipette.  This  is  to 
be  at  hand  when  the  abscess  is  opened  ;  both  ends  are  to  be 
broken  off  and  passed  through  the  flame  two  or  three  times.  The 
pipette  should  then  be  held  by  an  assistant,  or  put  with  the  end 
which  is  to  be  inserted  into  the  pus  projecting  over  the  side  of  the 
table,  so  as  not  to  come  into  contact  with  any  object. 

When  the  abscess  has  been  opened,  a  considerable  quantity  of 
pus  should  be  allowed  to  flow  out,  and  the  sterilized  pipette  is 
then  to  be  passed  through  the  incision  (care  being  taken  to  avoid 
contact  with  its  sides)  and  the  pus  carefully  sucked  up  into  the 
bulb.  The  fluid  thus  obtained  may  be  used  to  inoculate  cultures 
there  and  then,  or  both  ends  of  the  pipette  may  be  sealed  in  the 
flame. 

The  Examination  of  Pus. 

The  organisms  which  may  cause  pus  are  extremely  numerous, 
the  most  important  being  streptococci,  staphylococci,  the  pneumo- 
coccus  and  the  gonococcus,  the  bacilli  of  typhoid  fever,  tubercu- 
losis, and  glanders,  the  B.  coli  communis,  the  B.  pyocyaneus  (the 
organism  which  produces  blue  pus),  and  the  fungus  of  actino- 
mycosis. In  the  majority  of  cases  the  organism  which  is  present 
in  a  given  sample  of  pus  can  be  determined  by  a  microscopic 
examination  of  films  prepared  in  the  usual  way  and  stained  by  a 

1 02 


THE    COLLECTION    AND    EXAMINATION    OF    PUS  I63 

simple  stain,  such  as  carbol  thionin.  A  specimen  should  also  be 
stained  by  Gram's  method  and  the  results  compared. 

When  cultural  examinations  are  required,  the  best  plan  is  to 
make  stroke  cultivations  on  agar  in  the  manner  described  on 
p.  16,  and  incubate  them  for  twenty-four  hours  at  the  temperature" 
of  the  body.  The  appearances  of  the  colonies  will  be  similar  to 
those  described  as  occurring  in  cultures  made  from  the  blood,  to 
which  the  reader  is  referred.  It  is  to  be  noted,  however,  that  the 
gonococcus  will  not  grow  under  such  circumstances  unless  the 
surface  of  the  medium  has  previously  been  coated  with  blood. 

Another  method  is  to  make  gelatin  plates.  This  is  a  very 
simple  matter  if  the  materials  are  at  hand. 

Requisites. — i.  Two  or  three  tubes  of  gelatin. 

2.  Two  or  three  sterilized  Petri  dishes. 

3.  A  platinum  needle — a  loop  will  be  best. 

Process.—  Inoculate  a  gelatin  tube  in  the  manner  described  on 
p.  16,  and  then  melt  it  by  immersion  in  warm  (not  hot)  water. 

Distribute  the  pus  throughout  the  melted  gelatin  by  rolling  the 
tube  between  the  hands,  and  by  tilting  it  from  side  to  side.  Do  not 
shake,  and  do  not  let  the  melted  gelatin  touch  the  cotton-wool  plug. 

Take  a  loopful  of  the  gelatin  and  transfer  it  to  a  second  culture- 
tube.  Melt  the  gelatin  in  this  and  mix  as  before.  Proceed  to 
inoculate  a  third  tube  from  the  second  one  if  you  think  it  probable 
that  the  pus  is  very  rich  in  organisms. 

Now  take  the  first  tube  and  singe  the  projecting  part  of  the 
wool  plug,  and  heat  the  mouth  of  the  tube  in  order  to  destroy  any 
germs  which  may  be  upon  it ;  allow  it  to  cool. 

Place  the  Petri  dish  on  the  table  in  front  of  you,  and  raise  the 
lid  sufficiently  to  allow  you  to  insert  the  end  of  the  test-tube  ;  do 
this,  and  tilt  the  latter  so  that  the  melted  gelatin  flows  into  the 
dish.  Immediately  replace  the  lid,  and  tilt  and  roll  the  dish  until 
the  gelatin  forms  an  even  film  over  its  whole  lower  surface. 
Place  it  on  a  flat  table  to  set.  Repeat  the  process  with  the  other 
tubes.  Incubate  at  about  20°  C.  for  two  or  three  days.  Examine 
the  dishes,  placing  them  on  the  stage  of  the  microscope  and  using 
the  low  power.  Each  organism  will  have  grown  into  a  small 
colony,  which  will  resemble  those  which  are  described  in  the 
section  on  the  blood.  There  will  be  slight  differences,  but  not 
enough  to  lead  to  error  if  the  examination  of  the  colonies  is 
supplemented  by  an  inspection  of  stained  films. 

The  pneumococcus,  gonococcus,  the  fungus  of  actinomyces,  and 


104  CLINICAL    BACTERIOLOGY   AND    HEMATOLOGY 

the  tubercle  bacillus,  will  not  grow  on  these  plates ;  the  strepto- 
coccus and  the  bacillus  of  glanders  will  grow  feebly,  if  at  all. 

In  a  day  or  two  longer  the  plates  will,  in  some  cases,  be  found 
to  have  undergone  a  decided  change.  If  liquefying  organisms  are 
present  the  colonies  will  soon  become  depressed  below  the  general 
surface  of  the  medium,  and  will  be  surrounded  by  haloes  which 
consist  of  liquefied  gelatin.  This  will  happen  with  the  staphylo- 
cocci and  the  B.  pyocyaneus ;  not  with  the  streptococci,  the 
typhoid  bacillus,  nor  with  the  B.  coli. 

The  bacillus  of  blue  pus  can  readily  be  distinguished  from  the 
staphylococci  by  its  morphological  appearance  (it  is  a  slender  rod), 
and  by  the  fact  that  the  gelatin  round  the  colony  is  coloured  blue 
or  bluish-green,  the  growth  itself  being  nearly  white. 

Interpretation  of  Results. 

The  chief  practical  value  of  the  bacteriological  examination  of 
pus  is  derived  from  the  fact  that  if  specific  vaccine  treatment  (on 
Wright's  system)  is  to  be  used,  the  vaccine  must  be  prepared  from 
the  organism  which  is  causing  the  disease.  If  a  patient  is  suffer- 
ing from  a  staphylococcic  lesion,  it  is  not  much  use  inoculating  him 
for  pneumococci,  or  vice  versa.  I  have  seen  and  obtained  such 
excellent  results  in  some  cases  by  treatment  of  this  nature  that 
very  little  doubt  remains  in  my  mind  that  the  method  is  one  which 
will  be  widely  used  in  the  future.  Except  for  this  the  results 
obtained  by  a  study  of  the  bacteria  in  pus  are  more  of  scientific 
interest  than  of  practical  importance.  It  is  the  situation  of  the 
collection  of  pus  rather  than  the  bacteria  causing  it  which 
influences  treatment  and  prognosis.  A  list  of  the  more  important 
results  which  are  produced  by  the  chief  pyogenic  bacteria  may  be 
of  interest. 

Staphylococci  are  the  chief  producers  of  localized  suppuration  in 
the  skin — such,  for  instance,  as  that  which  occurs  in  boils, 
carbuncles,  impetigo,  folliculitis,  etc.  They  may  cause  abscesses 
in  any  part  of  the  body,  and  may  also  give  rise  to  general  infec- 
tions, ulcerative  endocarditis,  etc.,  though  this  is  rare. 

It  is  in  the  localized  skin  affections  of  staphylococcic  origin 
especially  that  good  results  are  obtainable  by  specific  vaccination, 
and  a  cure  may  often  be  obtained  in  cases  which  are  very  intract- 
able by  other  methods. 

Streptococci  usually  cause  spreading  inflammation  of  the  type  of 
erysipelas  or  cellulitis.     They  are  common  causes  of  osteomyelitis 


THE   COLLECTION   AND   EXAMINATION    OF   PUS  I05 

and  suppurative  and  septicaemic  or  pyaemic  processes  connected 
with  the  puerperium. 

In  general  infections  which  are  due  to  streptococci  the  use  of 
antistreptococcic  serum  is  indicated,  and  may  offer  the  only  hope 
for  the  patient. 

The  pneumococcus  often  produces  suppuration  in  connection  with 
the  respiratory  system,  especially  empyema.  It  also  causes  many 
cases  of  suppurative  otitis  media  and  meningitis. 

The  bacillus  of  typhoid  fever  sometimes  causes  abscesses  in  con- 
nection with  the  bones  after  (sometimes  long  after)  typhoid  fever. 
It  has  been  found  in  other  suppurative  conditions,  e.g.,  empyema. 

The  tubercle  bacillus  gives  rise  to  "cold  abscesses,"  usually  in 
connection  with  bone.  The  suppuration  which  occurs  in  the  walls 
of  phthisical  vomicae  is  due  to  other  bacteria,  chiefly  streptococci 
and  staphylococci.  The  pus  in  true  tuberculous  abscesses  is  thin 
and  watery,  like  milk  and  water,  and  often  contains  small  caseous 
masses.  The  cells  are  usually  mostly  lymphocytes.  In  most 
cases  it  is  perfectly  easy  to  find  tubercle  bacilli  in  this  pus  if  it  is 
examined  when  the  abscess  is  first  opened,  whereas  afterwards 
none  may  be  found  after  a  very  long  and  painstaking  search. 

The  bacillus  of  glanders  only  causes  suppuration  in  the  specific 
lesions  of  the  disease  when  these  run  an  acute  course. 

The  B.  coli  communis  is  the  chief  cause  of  suppuration  occurring 
in  connection  with  the  abdominal  viscera,  especially  of  peritonitis 
due  to  perforation  of  the  intestine  and  appendicitis.  It  frequently 
attacks  the  urinary  passages,  causing  cystitis,  etc. 

The  B.  pyocyaneus  causes  blue  pus,  usually  in  connection  with 
the  skin  or  subcutaneous  tissues. 

The  fungus  of  actinomycosis  has  been  dealt  with  already. 

THE  BACTERIOLOGICAL  EXAMINATION  OF  THE 
MOUTH  AND  FAUCES 

The  method  of  examination  of  the  morbid  products  of  the  mouth 
and  fauces   in   diphtheria  has  been  explained   in   full,  and   the 
methods  which  are  used  in  other  conditions  are  similar  in  nature. 
The  more  important  of  these  allied  conditions  are  : 
Simple  angina  and  follicular  tonsillitis. 
Vincent's  angina. 
Scarlatinal  angina. 
Thrush. 
Syphilitic  angina. 


I06  CLINICAL   BACTERIOLOGY   AND    HEMATOLOGY 

Methods. — An  examination  of  a  film  stained  by  a  simple  stain, 
and  of  a  second  prepared  by  Gram's  method,  is  usually  all  that 
is  necessary,  but  it  is  advisable  to  be  prepared  to  make  cultures 
subsequently  if  thought  requisite.  If  the  patient  is  seen  at  some 
distance  from  the  laboratory,  the  material  is  best  collected  on  a 
sterilized  swab  such  as  is  used  for  diphtheria  in  the  method  de- 
scribed on  p.  38,  taking  great  care  to  rub  it  on  the  affected  area.. 
When  this  is  brought  to  the  laboratory,  two  smears  are  to  be 
made  on  slides  which  have  just  been  sterilized  by  being  heated  in 
the  flame,  and  which  have  been  allowed  to  cool.  As  soon  as  the 
films  have  been  prepared  the  swab  is  to  be  returned  to  its  sterile 
tube  and  kept  in  readiness  for  the  preparation  of  cultures,  should 
they  be  required.  It  is  better  to  take  a  second  swab,  and  to  keep 
it  until  the  films  have  been  examined. 

When  the  patient  can  be  brought  to  the  laboratory  it  is  more 
convenient  to  collect  the  material  with  a  platinum  loop.  A  good 
loopful  of  the  material  is  removed,  laid  on  a  clean  slide,  and  two 
films  prepared  by  pressing  a  second  slide  firmly  on  the  first  and 
sliding  them  apart.  If  there  is  any  difference  between  the  two, 
the  thicker  is  used  for  staining  by  Gram's  method.  This  is  fixed 
in  the  flame  in  the  usual  way,  stained  by  aniline  gentian  violet 
(3  minutes),  rinsed,  fixed  in  Gram's  iodine  solution,  decolorized  in 
methylated  spirit  or  absolute  alcohol  until  no  more  colour  comes 
out,  stained  in  dilute  (i  in  5  or  i  in  10)  carbol  fuchsin  for  a  quarter 
of  a  minute,  washed,  and  dried.  The  other  film  is  best  stained  by 
carbol  thionin,  but  Loffler's  blue  answers  very  well. 

The  examination  of  the  films  is  made  at  once,  and  will  show 
whether  a  cultural  examination  is  necessary,  and  if  so,  what 
medium  should  be  used.  Thus,  if  Gram-staining  baciUi  are 
present,  diphtheria  is  suspected,  and  cultures  should  be  made  on 
blood-serum. 

Simple  Angina  and  Follicular  Tonsillitis  may  be  due  to 
streptococci,  staphylococci,  pneumococci,  or  the  Micrococcus  catar- 
rhalis.  These  are  readily  detected  in  the  smears,  the  first  as  longer 
or  shorter  chains  of  cocci,  the  second  as  cocci  which  are  isolated 
or  in  small  groups  and  often  contained  in  the  leucocytes,  the  third 
as  pairs  of  cocci  with  a  more  or  less  marked  lanceolate  shape 
and  a  capsule:  all  these  stain  by  Gram.  The  M.  catarvhalis 
(Plate  III.,  Fig.  5)  is  recognisable  by  its  shape  (kidney-shaped,  or 
a  sphere  with  a  segment  cut  off),  by  its  being  larger  in  size  than 
the  staphylococcus,  by  its  being  frequently  intracellular,  and  by 


BACTERIOLOGICAL  EXAMINATION  OF  MOUTH  AND  FAUCES     IO7 

its  not  staining  by  Gram's  method.     Here  a  warning  already  given 
must  be  repeated  :  it  is  not  safe  to  conclude  that  an  organism 
does   not  stain  by  Gram  because  the  intracellular  bacteria  are 
decolorized,  and  extracellular  ones  should  be  sought  for,  and  Ynll__ 
usually  be  found. 

If  no  organisms  but  the  above  cocci  are  found  after  a  very 
careful  search,  the  conclusion  is  that  the  case  is  probably  either 
simple  angina,  follicular  tonsillitis,  ulcerative  tonsillitis,  or  scarlet 
fever.  But  it  must  be  remembered  that,  although  a  good  large 
area  of  film  has  been  searched,  in  reality  but  a  very  small  volume 
of  secretion  has  come  under  observation — probably  less  than  a 
cubic  millimetre — and  that  diphtheria  bacilli  may  be  present ;  it 
is  therefore  advisable  to  make  a  culture  on  blood-serum  before 
giving  a  definite  diagnosis. 

No  conclusions  as  to  the  cause  of  the  angina  or  as  to  its  prog- 
nosis can  be  given  from  the  discovery  of  the  causal  organism. 
The  presence  of  a  streptococcus  renders  it  more  likely  that  the 
disease  is  scarlet  fever,  but  certainly  does  not  prove  it.  Nor  does 
the  examination  give  much  help  as  to  treatment.  If  the  disease  is 
due  to  a  streptococcus  and  the  symptoms  are  severe,  antistrepto- 
coccic serum  may  be  given,  the  local  treatment  being  continued 
as  usual. 

Vincent's  Angina  is  a  very  interesting  form  of  sore  throat 
recently  described  by  Professor  Vincent,  of  Paris,  and  is  especially 
important  since  (i)  it  closely  resembles  diphtheria,  and  the  two  may 
be  readily  confounded,  and  it  also  may  easily  be  confused  with  a 
syphilitic  lesion;  and  (2)  it  is  readily  cured  by  appropriate  treat- 
ment— friction  twice  daily  with  a  tampon  soaked  in  tincture  of 
iodine.  It  has  attracted  very  little  attention  in  this  country,  yet 
it  does  not  seem  to  be  rare ;  I  have  now  seen  many  cases. 
One  was  of  great  severity,  and  was  associated  with  the  forma- 
tion of  an  abscess  in  the  tonsil,  and  subsequently  of  another 
in  the  soft  palate. 

Vincent  describes  two  forms  : 

I .  An  ulcero-membranous  variety,  which  commences  with  fever 
and  general  malaise,  and  with  redness  of  a  tonsil  or  of  a  pillar  of 
the  fauces.  In  a  day  or  two  a  grey  or  yellowish  false  membrane 
appears  on  the  injected  area ;  it  is  soft  and  but  slightly  adherent, 
and  when  removed  the  mucous  membrane  is  found  to  be  ulcerated. 
As  the  disease  proceeds  the  membrane  increases  in  thickness,  and 
a   deep  ulcer  is  formed.     The  breath   is  foetid  and  the  tongue 


I08  CLINICAL    BACTERIOLOGY   AND    HEMATOLOGY 

furred,  salivation  occurs,  and  deglutition  is  painful.  The  sub- 
maxillary glands  may  be  enlarged.  In  most  cases  the  patient 
recovers  in  a  week  or  fortnight,  but  the  affection  may  become 
chronic  and  last  a  month  or  more. 

More  severe  forms  occur  in  which  the  soft  palate,  uvula, 
tongue,  etc.,  are  invaded,  and  ulceration  also  occurs.  In  some 
cases  there  is  a  scarlatiniform  rash,  which  might  lead  to  the 
diagnosis  of  scarlet  fever. 

2.  The  diphtheroid  form  is  rarer,  occurring  only  in  2  per  cent, 
of  Vincent's  cases.  The  onset  is  accompanied  by  a  little  fever, 
some  difficulty  in  swallowing,  and  foetor  of  the  breath.  Locally, 
the  mucous  membrane  is  inflamed  and  injected,  and  a  whitish 
membrane  is  formed ;  it  is  thin  at  first,  but  becomes  thicker,  and 
when  removed  leaves  an  ulcerating  or  bleeding  surface,  but  the 
ulceration  is  less  than  in  the  other  form.  The  disease  runs  a 
shorter  course,  recovery  occurring  in  four  to  eight  days.  This  is 
the  form  which  so  closely  resembles  diphtheria,  and  which,  I  have 
no  doubt,  has  often  been  mistaken  for  it,  even  after  a  superficial 
bacteriological  examination  of  the  membrane. 

The  diagnosis  is  made  from  films  prepared  from  the  swabs, 
stained  by  Gram's  method  and  counterstained  by  carbol  fuchsin. 
In  the  more  common  form  (the  ulcero-membranous)  two  very 
interesting  organisms  will  be  found — a  bacillus  and  a  spirillum 
(Plate  IV.,  Pig.  2).''  In  the  diphtheroid  form  the  bacillus  is 
present,  but  not  the  spirillum  ;  in  either  form  of  the  disease  the 
characteristic  organism  or  organisms  may  be  associated  with 
streptococci,  staphylococci,  etc.,  and  these  secondary  infections 
may  give  rise  to  grave  complications. 

The  B.  fasiformis,  which  occurs  in  both  forms  of  the  disease, 
may  be  found,  in  small  numbers,  as  a  normal  inhabitant  of 
the  mouth,  and  occurs  in  myriads  in  the  disease.  It  varies  in 
size,  but  is  on  the  whole  a  large  bacillus,  about  as  long  as  the 
diameter  of  a  red  blood-corpuscle,  or  even  longer.  Typically  it 
has  both  ends  pointed,  giving  it  the  shape  of  a  greatly  elongated 
spindle,  but  other  forms  always  occur,  and  may  even  constitute 
the  majority  of  the  bacilli  present.  It  often  contains  two  or  three 
clear  vacuoles,  which  may  not  be  noticed  if  the  staining  is  too 
deep.  Both  the  bacillus  and  the  spirillum  are  usually  actively 
motile,  and  it  is  a  good  plan  to  check  the  results  of  the  examina- 

*  It  is  possible  that  these  are  different  stages  of  the  same  organism,  a 
protozoon. 


BACTERIOLOGICAL  EXAMINATION  OF  MOUTH  AND  FAUCES      lOQ 

tion  of  the  stained  films  by  mounting  a  fresh  wet  specimen 
between  slide  and  cover-glass,  and  examining  it  under  the  oil- 
immersion  lens. 

The  B.  fusiformis  plays  a  very  important  part  in  many  in- 
flammatory and  ulcerative  conditions  in  and  about  the  mouth 
and  adjacent  cavities,  the  teeth,  etc.  For  instance,  in  association 
with  the  same  spirillum  it  is  present  in  the  pus  of  pyorrhoea 
alveolaris.  The  lesions  it  causes  are  all  associated  with  a  foetid 
odour,  and  the  cultures  (which  are  very  difficult  to  obtain)  have  a 
similar  smell. 

The  spirillum  occurs  only  in  the  ulcero- membranous  form, 
and  is  present  in  vast  numbers,  usually  even  more  plentifully 
than  the  bacillus ;  a  well-prepared  specimen  is  one  of  the  most 
striking  and  characteristic  objects  to  be  seen  in  the  whole  range 
of  bacteriology.  It  is  much  longer  than  the  bacillus,  very  thin, 
and  either  wavy  and  irregular  in  outline  or  thrown  into  definite 
corkscrew  curves.  These  are  better  seen  in  a  wet  specimen, 
though  here  the  active  motility  of  the  organism  often  makes  it 
impossible  to  make  out  its  exact  shape.  It  usually  stains  badly, 
and  I  have  missed  it  in  specimens  rapidly  stained  with  weak 
stains  :  dilute  carbol  fuchsin  stains  it  very  well  in  a  quarter  of  a 
minute.  In  one  or  two  specimens  I  found  the  spirilla  broken  up 
into  chains  of  very  minute  granules,  so  that  they  resembled  long 
chains  of  very  minute  streptococci.  In  each  case  it  was  late  in 
the  disease,  so  that  they  may  have  been  degeneration  forms. 

The  spirillum  differs  from  that  of  syphilis  in  that  it  is  much 
larger  and  is  easily  stained  by  ordinary  dyes,  and  the  two  could 
hardly  be  mistaken.  It  is  to  be  noted,  however,  that  the 
B.  fusiformis  is  not  uncommon  in  syphilitic  lesions. 

Scarlatinal  Angina  cannot  be  diagnosed  with  certainty  from 
other  forms  of  sore  throat  by  bacteriological  methods.  It  is  usually 
due  to  a  streptococcus  which  occurs  in  very  long  chains  and  is 
somewhat  characteristic,  but  which  cannot  be  diflferentiated  from 
other  forms  of  streptococci  by  simple  means. 

Thrush  is  usually  easily  recognisable,  but  when  this  is  not  the 
case  a  Gram-stained  specimen  of  the  membrane  will  immediately 
settle  the  diagnosis.  The  specific  organism,  the  Oidium  albicans 
or  Saccharomyces  albicans,  is  a  mould  which  appears  in  the  form 
of  large  and  thick  branching  mycelial  filaments  which  stain  deeply 
by  Gram's  method,  and  which  are  interspersed  with  large  round 
or  oval  spores,  which  also  stain  readily  and  deeply.    The  organism 


no  CLINICAL    BACTERIOLOGY    AND    HEMATOLOGY 

can  be  readily  diflferentiated  from  the  bacterial  filaments  which  may 
occur  in  the  mouth  (leptothrix,  etc.)  by  its  relatively  enormous  size. 
When  cultivated  for  a  day  or  two  on  ordinary  media  mycelium 
formation  does  not  take  place,  or  only  to  a  very  limited  extent, 
and  the  oval  or  spherical  spores  are  often  mistaken  for  yeasts. 

Syphilitic  Angina  may  be  recognised  by  the  identification  of 
the  Spirillum  pallida^  but  some  caution  is  necessary,  since  non- 
pathogenic spirilla  are  frequently  present  in  the  healthy  mouth. 
The  films  stained  by  Giemsa's  stain  must  be  very  carefully 
compared  with  others  stained  by  simple  dyes  (thionin  or  methylene 
blue),  to  make  sure  that  the  organism  found  is  not  coloured  by 
ordinary  means. 

THE  BACTERIOLOGICAL  EXAMINATION  OF  THE 
NOSE  AND  ACCESSORY  CAVITIES 

In  health  the  nasal  mucous  membrane  is  sterile  except  for  that 
portion  in  close  proximity  to  the  orifices ;  the  vibrissae  are 
especially  contaminated  with  air-borne  organisms,  and  contact 
with  them  must  be  avoided  if  cultures  are  being  taken. 

Methods. — In  most  cases  a  simple  microscopical  examination  of 
the  mucus,  muco-pus,  or  pus  from  the  nose  is  sufficient,  and  the 
material  may  be  taken  from  the  patient's  pocket-handkerchief 
immediately  after  he  has  blown  his  nose.  Where  cultures  are 
required  the  methods  are  more  difficult,  and  the  material  must  be 
collected  as  near  as  possible  to  the  region  where  it  is  secreted ; 
this  is  especially  the  case  in  the  examination  of  the  pus  from 
cases  of  empyema  of  the  antrum,  frontal  sinus,  etc.  I  have 
found  the  most  convenient  instrument  is  a  long  capillary  pipette 
of  rather  wide  calibre,  and  bent  to  an  angle  of  about  135  degrees, 
at  a  point  some  4  inches  from  its  tip,  which  must  be  carefully 
rounded  in  the  flame,  so  as  not  to  injure  the  mucous  membrane. 
It  is  provided  with  an  indiarubber  nipple,  and  is  readily  prepared 
from  one  of  the  straight  pipettes  described  on  p.  151,  by  heating 
it  gently  in  a  spirit-lamp,  at  a  point  about  4  inches  from  the  tip, 
until  the  glass  is  just  softened,  and  then  allowing  the  distal  end 
to  fall  until  the  proper  angle  is  reached.  The  pipette  may  be 
sterilized  by  passing  it  rapidly  through  the  flame,  taking  care  not 
to  melt  it. 

To  use  it,  seat  yourself  opposite  to  the  patient,  insert  a  nasal 
speculum  sterilized  in  carbolic  acid  or  in  the  flame,  and  examine 
the   nose  in  the   ordinary  way   by   means   of  a   beam   of  light 


BACTERIOLOGICAL   EXAMINATION    OF   THE   NOSE,    ETC.    Ill 

directed  up  the  nose  from  a  laryngoscope  mirror.  Insert  the  tip 
of  the  pipette,  taking  great  care  not  to  touch  the  vibrissae  or  the 
mucous  membrane  near  the  orifice,  and  pass  it  upward  until  it 
comes  in  contact  with  the  pus.  Then  squeeze  the  nipple  and 
allow  it  to  expand  again,  slightly  moving  the  tip  of  the  pipette 
about  in  the  pus,  and  taking  care  not  to  bring  it  in  contact  with  the 
mucous  membrane.  In  most  cases  you  will  be  able  to  suck  up 
a  few  drops  of  pus  ;  sometimes  it  is  very  thick  and  turbid,  and  will 
not  enter  the  pipette,  and  in  this  case  you  must  use  a  platinum 
loop,  which  is  not  nearly  so  efficacious  or  so  easy  to  use.     But  in 


Fig.  29. — Angled  Pipette  for  Collecting  Pus  from  the  Nose. 


most  cases  the  angled  pipette  will  be  found  available  after  a  very 
little  practice. 

The  organisms  most  frequently  found  in  inflammation  of  the 
general  surface  of  the  mucous  membrane  of  the  nose  are  the 
diphtheria  bacillus,  the  influenza  bacillus,  the  M.  catarvhalis, 
and,  less  commonly,  the  pneumococcus.  These  do  not  call  for 
further  mention.  In  early  cases  of  leprosy  the  bacilli  may  be 
found  in  the  nose,  apparently  before  they  are  present  elsewhere 
in  the  body,  and  the  diagnosis  may  be  made  from  their  recognition. 

In  suppuration  of  the  antrum  and  other  sinuses  the  bacteriology 
is  very  variable.  In  disease  of  the  antrum  due  to  carious  teeth 
the  B.  fusiformis^  mixed  with  numerous  other  organisms,  may  be 
found.  In  other  cases  the  commonest  bacteria  are  pneumococci, 
streptococci,  staphylococci,  and  the  M.  catarrhalis,  usually  unmixed 
in  each  case  ;  the  subject,  however,  has  not  yet  been  sufficiently 
investigated. 

THE  CONJUNCTIVA 

The  method  of  examination  is  very  simple.  A  little  pus  is  taken 
with  a  platinum  loop  and  films  prepared.  One  of  these  is  stained 
with  carbol  thionin  or  Loffler's  blue,  and  the  other  by  Gram 
followed  by  dilute  carbol  fuchsin. 


112  CLINICAL   BACTERIOLOGY   AND    HEMATOLOGY 

The  most  common  causes  of  conjunctivitis  are  the  gonococcus, 
the  pneumococcus,  the  Koch-Weeks  bacillus,  and  the  bacillus  of 
Morax  and  Axenfeld.  In  addition  to  these,  certain  other  bacteria 
must  be  briefly  mentioned. 

The  method  for  the  recognition  of  the  gonococcus  need  not  be 
further  described  (see  p.  84),  nor  is  it  necessary  to  point  out  the 
danger  of  this  form  of  conjunctivitis,  nor  the  fact  that  prompt 
measures  must  be  taken  if  the  eye  is  to  be  saved. 

Other  organisms  somewhat  resembling  the  gonococcus  some- 
times occur  in  the  conjunctiva,  but  I  have  never  seen  or  heard  of 
a  case  in  which  they  were  absolutely  identical  in  appearance  and 
distribution,  and  present  in  numbers  sufficient  to  lead  to  errors  in 
diagnosis. 

The  pneumococcus  is  not  a  common  cause  of  conjunctivitis,  and 
when  it  occurs  the  prognosis  with  regard  to  the  involvement  of 
the  cornea  is  not  serious. 

The  bacillus  of  Koch  and  Weeks  is  extremely  minute,  and  has 
a  close  resemblance  to  the  influenza  bacillus.  It  is  very  thin  in 
proportion  to  its  length,  does  not  stain  by  Gram,  and  is  fre- 
quently intracellular,  the  cells  then  containing  large  numbers  of 
bacilli,  though  it  may  be  necessary  to  search  over  a  considerable 
area  of  film  before  an  aff"ected  cell  is  seen.  It  is  advisable  to 
search  for  the  organisms  in  a  thionin  or  Lofller's  blue  specimen, 
as  it  will  probably  be  more  distinct  than  in  the  Gram  specimen 
counterstained  with  carbol  thionin. 

If  the  two  occurred  in  the  same  region,  it  would  probably  be 
indistinguishable  from  the  influenza  bacillus ;  they  differ,  how- 
ever, in  cultural  characters. 

It  causes  the  common  self-limited  variety  of  acute  or  chronic 
conjunctivitis.  There  is  no  danger  that  the  cornea  may  become 
infected  ;  the  disease  is  very  contagious. 

The  bacillus  of  Morax  and  Axenfeld  (Plate  IV.,  Fig.  3)  causes  the 
dry  conjunctivitis  which  occurs  especially  along  the  edges  of  the 
eyelids  and  at  the  angles  of  the  eye,  and  which  does  not  tend  to 
cure  in  the  absence  of  appropriate  treatment.  The  secretion  is 
usually  very  scanty,  and  not  purulent ;  it  is  best  collected  from 
the  caruncle,  and  it  may  be  necessary  to  use  a  capillary  pipette 
for  the  purpose. 

It  is  readily  recognised  as  a  rather  large,  broad  bacillus,  with 
the  sides  parallel  and  the  angles  slightly  rounded,  and  two  bacilli 
are  often  seen  with  their  ends  approximated  together.     It  is  most 


THE    SPUTUM  113 

frequently  extracellular,  but  some  bacilli  are  frequently  seen  within 
the  cells.  Gram's  stain  is  not  retained.  It  grows  readily  on  blood- 
serum,  which  is  rapidly  liquefied. 

Of  the  rarer  causes  of  conjunctivitis,  the  staphylococcus,  strepto- 
coccus, and  pneumobacillus  may  be  mentioned.  The  latter  may 
be  recognised  by  the  presence  of  a  capsule  surrounding  a  bacillus 
which  greatly  resembles  an  elongated  pneumococcus,  which  does 
not  retain  Gram  (Plate  III.,  Fig.  3). 

Diphtheria  affects  the  conjunctiva,  and  its  clinical  recognition  is 
not  usually  difficult.  But  the  bacteriological  diagnosis  (without 
the  use  of  animal  inoculations)  is  complicated  by  the  fact  that 
an  organism — the  xerosis  bacillus — which  closely  resembles  the 
diphtheria  bacillus  may  occur  in  the  conjunctiva  either  in  health 
or  in  disease.  It  does  not  appear  that  the  two  can  be  differen- 
tiated with  certainty  by  morphological  appearances,  staining 
reactions  (the  xerosis  bacillus  ol'ten  shows  polar  granules),  or 
even  by  cultural  tests. 

In  tuberculosis  of  the  conjunctiva  it  is  usually  necessary  to  excise 
a  piece  of  the  lesion,  and  cut  sections,  but  bacilli  have  been 
detected  occasionally  in  scrapings. 

THE  SPUTUM 

The  chief  applications  of  bacteriology  to  the  examination  of  the 
sputum  have  been  mentioned  already,  but  it  seems  advisable  to 
add  a  few  general  words  on  the  subject. 

The  selection  of  the  material  is  of  prime  importance,  since  it 
is  obviously  useless  to  expect  an  examination  of  the  secretion 
of  the  mouth  and  fauces  to  yield  information  as  to  the  state  of 
the  lungs.  Where  the  sputum  is  copious  the  danger  of  this  error 
is  not  great ;  in  advanced  phthisis  the  contents  of  a  sputum-cup 
taken  at  random  will  usually  show  signs  of  tubercle  bacilli.  Where 
the  sputum  is  but  scanty  the  possibility  is  great,  and  in  this  case 
the  patient  should  be  supplied  with  a  clean  (and,  if  possible,  sterile) 
sputum-cup,  to  be  used  only  for  collecting  the  material  for  examina- 
tion, and  he  must  be  instructed  to  use  it  only  after  a  paroxysm 
of  coughing,  and  when  he  distinctly  feels  the  sputum  come  up  from 
the  chest.  If  the  patient  is  not  sufficiently  intelligent,  the  task  of 
collecting  the  sputum  may  be  entrusted  to  a  nurse,  to  whom  the 
importance  of  securing  material  directly  from  the  lungs  has  been 
explained. 

8 


1 1  4  CLINICAL    BACTERIOLOGY   AND    HEMATOLOGY 

Where  it  is  necessary  to  work  with  material  which  has  been 
collected  without  any  precaution,  the  microscopical  nature  of 
the  cells  may  afford  a  clue  as  to  its  origin.  Mucus  or  muco-pus 
derived  from  the  mouth,  pharynx,  etc.,  is  characterized  by  containing 
squamous  cells  ;  these  are  of  large  size,  flattened,  have  a  compara- 
tively small  nucleus,  and  are  often  collected  into  groups  of  three 
or  four,  with  a  distinct  tessellated  arrangement.  They  contain 
granules  (of  keratin  or  an  allied  substance)  which  stain  deeply  by 
Gram's  method  ;  they  are  also  the  last  substances  (other  than 
acid-fast  bacilli)  to  be  decolorized  by  the  acid  in  the  Ziehl-Neelsen 
process,  and  when  the  decolorization  has  not  been  carried  out 
quite  completely,  may  remain  stained  in  the  form  of  pinkish 
plaques.  The  sputum  which  comes  from  the  bronchi  may  be 
characterized  by  the  presence  of  columnar  cells,  which  are  occa- 
sionally found  to  be  ciliated,  but  in  most  cases  this  sign  fails,  and 
the  sputum  consists  of  mucus  enclosing  polynuclear  leucocytes 
and  no  characteristic  cells  of  any  kind.  The  same  is  true  of  the 
sputum  from  "the  lungs :  there  is  frequently  nothing  to  dis- 
tinguish it  from  that  of  other  regions,  and  this  is  especially  the 
case  when  it  is  derived  from  a  cavity  which  is  lined  with  pyogenic 
membrane.  But  sometimes  the  sputum  contains  very  charac- 
teristic alveolar  epithelial  cells.  These  are  derived  from  the  lining 
of  the  alveoli,  the  cells  of  which  lose  their  flattened  shape  and 
become  spherical  in  many  pathological  conditions.  They  are  large 
round  or  oval  cells,  much  larger  than  the  polynuclear  leucocytes, 
and  have  clear  protoplasm  and  a  round  or  oval  nucleus,  which  is 
often  placed  eccentrically.  They  are  actively  phagocytic,  and 
their  contents  give  an  important  clue  to  the  nature  of  the  patho- 
logical process  at  work  in  the  lungs,  since  they  are  derived  directly 
from  the  alveoli.  In  pneumonia  they  may  be  seen  to  contain 
pneumococci,  and  this  is  especially  the  case  after  the  crisis ;  in 
congestion,  and  especially  passive  congestion,  of  the  lung  they  are 
packed  with  red  corpuscles,  often  in  various  stages  of  destruction, 
or  with  granules  or  crystals  of  altered  blood-pigment.  In  diseases 
due  to  dust  the  cells  contain  fragments  of  the  dust  in  question, 
but  it  must  be  remembered  that  particles  of  coal-dust  are  often 
found  in  them  in  normal  conditions  in  city  dwellers. 

In  asthma  the  characteristic  Curschmann's  spirals  may  be  seen, 
and  the  cells  will  be  found  to  consist  almost  entirely  of  eosinophile 
leucocytes. 


THE   GASTRIC   CONTENTS    AND   VOMIT  II5 


THE  GASTRIC  CONTENTS  AND  VOMIT 

The  bacteriological  examination  of  the  stomach  contents  is  not 
of  much  importance  except  in  one  case — i.e.,  in  the  differential 
diagnosis  of  carcinoma  ventriculi  and  simple  dilatation  of  the 
stomach.  For  this  purpose  it  is  usually  sufficient  to  examine  the 
vomit,  or,  if  vomiting  does  not  occur,  the  gastric  contents  removed 
by  a  stomach- tube.  It  is  quite  unnecessary  to  give  a  test  meal, 
though  this  is  advisable  if  a  chemical  examination  is  to  be  made ; 
this  should  be  done  in  all  doubtful  cases,  but  since  the  methods 
requisite  are  outside  the  scope  of  this  work,  only  a  couple  of 
simple  tests  will  be  given. 

The  examination  is  carried  out  quite  simply  by  the  wet  method. 
Two  or  three  drops  of  the  vomit  (avoiding  undigested  food  or 
mucus,  since  the  latter  is  likely  to  be  derived  from  the  mouth  or 
pharynx)  are  placed  on  a  slide,  and  either  examined  just  as  they 
are,  or  a  drop  of  watery  methylene  blue,  gentian  violet,  or  other 
stain,  is  added  and  stirred  well  in,  and  the  mixture  covered  with  a 
cover-glass.  If  there  is  an  excess  of  fluid  (so  that  the  cover-glass 
is  floated  up),  it  may  be  sucked  up  by  means  of  a  piece  of  blotting- 
paper;  and  if  an  oil-immersion  lens  is  to  be  used,  it  will  be 
advantageous  to  seal  the  cover-glass  down  by  means  of  melted 
paraffin  applied  by  a  hot  iron,  so  as  to  prevent  it  from  being 
lifted  up  by  the  suction  of  the  oil  between  the  lens  and  cover- 
glass.  If  no  positive  results  are  found  in  the  first  specimen 
examined,  two  or  three  more  should  be  made,  as  the  characteristic 
organisms  are  often  not  distributed  uniformly  throughout  the 
vomit.  Subsequently  it  may  be  necessary  to  prepare  dried  films 
to  determine  whether  the  organisms  stain  by  Gram. 

In  carcinoma  of  the  stomach  the  characteristic  features  are 
(i)  the  presence  of  the  Boas-Oppler  bacillus,  (2)  the  usual  absence 
of  sarcinae,  and  (3)  the  absence  of  hydrochloric  and  presence  of 
lactic  acid.  In  addition  there  may  be  blood,  pus,  and  fragments 
of  tumour ;  the  latter  are  very  rare  and  difficult  to  diagnose  with 
certainty. 

The  Boas-OppUv  bacillus,  or  B.  geniculatuSf  is  very  characteristic 
of  carcinoma  of  the  stomach,  though  it  does  not  occur  in  every 
case.  It  is  very  rare  in  other  conditions ;  I  have  seen  it  once  in 
a  case  of  simple  chronic  gastritis,  but  here  it  was  present  in 
numbers  which  were  very  scanty  as  compared  with  the  profusion 

8—2 


Il6  CLINICAL   BACTERIOLOGY   AND    HEMATOLOGY 

in  which  it  occurs  in  malignant  disease,  and  it  would  have  been 
overlooked  on  a  casual  examination.  It  is  a  bacillus  of  large  size, 
and  has  a  tendency  to  grow  into  long  threads,  which  are  readily 
visible  under  a  J-inch  lens  (Plate  IV.,  Fig.  5).  In  a  wet  specimen 
these  threads  usually  seem  continuous,  but  on  examining  a 
dried  and  stained  specimen,  they  may  be  seen  to  be  composed  of 
bacillary  segments,  much  like  the  chains  of  the  anthrax  bacillus. 
In  a  wet  unstained  specimen  these  threads  are  very  easily 
recognised,  as  they  are  highly  refractile  and  may  have  a  slow, 
crawling  motion,  or  may  be  non-motile :  most  usually  the  latter. 
Another  characteristic  feature  is  the  presence  of  an  obtuse  angle 
in  some  portion  of  the  length  of  many  of  the  threads,  whence  the 
name  B.  geniculatus  is  derived.  The  organism  does  not  form 
spores.  It  stains  by  Gram,  and  I  have  noted  in  several  cases  the 
presence  of  a  phenomenon  which  is  very  rare  amongst  bacilli, 
that  of  longitudinal  "fission  in  a  small  number  of  the  chains, 
which  thus  come  to  form  double  rows  of  bacilli  in  close  lateral 
approximation.  That  this  is  not  a  mere  effect  of  staining  is 
shown  by  the  fact  that  occasionally  the  two  bacilli  at  one  end  of 
this  double  thread  will  turn  away  from  one  another,  so  that  it 
looks  as  if  there  were  true  branching  at  the  extremity  of  the 
filament.  If  this  turns  out  to  be  the  case  in  all  specimens,  it  will 
be  a  most  important  means  of  identifying  the  organism.* 

Cultures  may  readily  be  obtained  on  media  rather  highly 
acidified  with  lactic  acid,  but  are  not  requisite  for  the  identifica- 
tion of  the  bacillus. 

In  addition  to  the  Boas-Oppler  bacillus,  the  vomit  may  contain 
a  few  yeasts,  distinguished  by  their  large  size,  oval  or  spherical 
shape,  and  by  the  way  in  which  they  reproduce  themselves  by 
budding ;  there  may  also  be  a  few  bacteria  of  other  sorts.  But 
in  many  cases  the  bacilli  are  present  in  a  state  of  almost  absolute 
purity,  and  in  large  numbers,  so  that  they  form  tangled  masses. 

In  nearly  all  cases  in  which  the  bacillus  is  found  the  vomit 
contains  no  hydrochloric  acid,  or  only  a  trace.  To  test  for  it 
filter  some  of  the  vomit  and  place  a  drop  or  two  of  the  filtrate  on 
a  white  porcelain  tile ;  place  a  drop  of  a  0*5  per  cent,  alcoholic 
solution  of  dimethyl-amido-azo-benzol  close  to  it  and  let  the  two 
gradually  mix.  If  physiologically  active  HCl  is  present,  even  in 
very  small  amount,  a  transient  pink  colour  will  be  produced.    The 

*  Note  to  Third  Edition.  It  is  not,  though  I  have  since  seen  it  two  or 
three  times. 


THE   GASTRIC   CONTENTS    AND   VOMIT  II7 

commonly  recommended  phloroglucin  and  vanillin  test  does  not 
demonstrate  the  presence  of  HCl  in  combination  with  proteids, 
and  since  this  is  usually  the  form  in  which  it  occurs  in  the 
stomach  contents  the  failure  to  obtain  the  phloroglucin  and 
vanillin  reaction  is  not  of  the  slightest  importance  in  diagnosis. 
The  use  of  the  latter  test  is,  I  think,  one  of  the  chief  reasons  for 
the  common  idea  that  the  absence  of  HCl  is  of  very  little  value 
in  the  diagnosis  of  malignant  disease.  Using  the  dimethyl-amido- 
azo-benzol  test,  I  have  rarely  found  it  fail,  and  in  the  only  case  I 
remember  in  which  the  acid  was  found  present  in  considerable 
amount  the  growth  was  found  at  operation  to  be  spreading  out- 
wards, and  hardly  to  involve  the  mucous  membrane  at  all.  But 
these  tests  were  mostly  made  on  the  fluid  obtained  after  a  test 
meal,  and  not  on  vomits. 

Lactic  acid  usually  occurs  in  the  absence  of  free  hydrochloric 
in  cancer  of  the  stomach,  but  its  presence  is  in  itself  a  fact  of 
little  value,  since  it  occurs  in  other  conditions.  To  test  for  it  in  a 
vomit  it  is  necessary  to  extract  the  acid  by  thoroughly  shaking  up 
some  of  the  fluid  with  ether,  pipetting  off  the  latter,  and  then 
allowing  it  to  evaporate.  The  acid  is  contained  in  a  state  of  com- 
parative purity  in  the  residue,  which  is  then  dissolved  in  water  and 
tested  in  the  following  way  ;  To  half  a  test-tubeful  of  i  in  40 
carbolic  add  one  or  two  drops  of  liq.  ferri  perchlor.  A  fine 
amethyst  colour  will  result,  and  will  be  changed  to  a  bright 
canary-yellow  on  the  addition  of  the  solution  of  the  ethereal 
extract,  if  the  latter  contains  lactic  acid.  It  is  not  sufficient  to 
apply  this  test  to  the  filtrate  of  a  vomit  direct,  although  this  is 
permissible  in  the  case  of  the  fluid  removed  from  the  stomach 
after  a  test  meal. 

The  vomit  in  cases  of  simple  dilatation  of  the  stomach  usually 
contains  a  variety  of  bacteria,  yeasts,  etc.,  but  the  most  character- 
istic organisms  are  the  sarcinae,  a  group  of  cocci  which  have 
very  definite  microscopical  characters.  They  have  the  property 
of  dividing  by  three  successive  divisions  in  the  three  planes  of 
space  (at  right  angles),  and  the  eight  resulting  cocci  do  not  com- 
pletely separate  from  one  another.  The  result  is  the  formation  of 
a  group  of  eight  cocci  which  form  one  mass,  having  the  shape  of 
a  bale  of  soft  material  tied  round  by  three  tightly  drawn  cords 
at  right  angles  to  one  another.  Successive  divisions  take  place 
parallel  to  these,  and  a  very  complex  colony  results.  The  sarcinae, 
as  a  rule,  are  decidedly  larger  than  ordinary  cocci,  though  not  as 


ri8  CLINICAL    BACTERIOLOGY   AND    HEMATOLOGY 

large  as  yeasts  ;  the  different  cocci  of  each  group  usually  vary  in 
size  amongst  themselves,  the  younger  forms  being  the  larger. 
Most  of  them  stain  by  Gram,  but  this  test  is  hardly  necessary, 
as  they  may  be  readily  recognised  in  unstained  wet  prepara- 
tions. 

They  occur  in  profusion  (often  mixed  with  yeasts)  in  many 
cases  of  simple  dilatation  of  the  stomach,  though  not  in  all. 
They  occur  in  other  conditions,  but  are  very  rarely  found  in  cases 
of  carcinoma,  and  this  is  the  only  importance  attaching  to  them. 


THE  URINE 

The  more  important  investigations  in  which  the  urine  has  to 
be  examined  have  been  mentioned  already.  See  p.  62  (tubercle 
bacilli)  and  p.  87  (gonococci). 

Methods. — Where  the  examination  is  to  be  microscopical  and 
not  cultural  it  is  not  usually  necessary  to  use  a  catheter  specimen. 

The  urine  is  to  be  passed  directly  into  a  sterilized  vessel  (or  at 
least  a  clean  and  dry  one),  the  first  portion  being  passed  into 
another  vessel  and  rejected. 

The  examination  should  commence  by  the  inspection  of  a 
hanging-drop  preparation,  first  under  the  \,  then  under  the  y\r. 
This  will  enable  you  to  recognise  the  presence  of  motile  bacilli, 
streptococci,  other  cocci,  etc. ;  pus,  blood,  epithelial  cells,  etc., 
and  crystals. 

In  most  cases  it  is  not  absolutely  necessary  to  centrifugalize 
the  urine,  since  the  bacteria  are  commonly  present  in  large 
numbers,  but  it  is  an  advantage  when  this  can  be  done.  It  is 
not  advisable  to  allow  the  urine  to  deposit  spontaneously,  as  there 
are  many  chances  of  accidental  contamination,  and  many  common 
bacteria  grow  with  great  rapidity  in  urine. 

Then  pass  on  to  film  preparations,  preferably  from  the  centri- 
fugalized  deposit.  Prepare  films  in  the  ordinary  way  and  stain  by 
Gram,  counterstaining  by  dilute  carbol  fuchsin.  This  will  enable 
you  to  study  the  organisms  more  closely,  and  to  see  whether 
they  retain  Gram  or  not :  most  of  them  do.  The  staphylococcus 
(which  appears  in  the  urine  mostly  as  diplococci)  and  the  M.  uvece 
do  so,  and  the  gonococcus  and  the  gonococcus-like  diplococcus 
which  causes  cystitis  are  decolorized. 

Where  cultures  are  required,  a  catheter  specimen  must  be  used. 


THE    URINE  iig 

The  catheter  must  be  boiled,  and  the  urinary  meatus  sterilized. 
The  first  portion  of  the  urine  is  to  be  rejected,  and  a  small 
quantity  of  the  last  part  collected  in  a  sterile  test-tube,  and 
the  plug  immediately  replaced.  A  small  quantity  only  is  re- 
quired. ^  . 

Cystitis,  Pyelitis,  Etc. — These  may  be  due  to  many  organisms, 
either  pure  or  mixed,  and  there  is  but  little  practical  interest  in 
their  recognition,  except  in  cases  in  which  the  vaccination  treat- 
ment is  to  be  tried.  There  is,  unfortunately,  no  method  of 
distinguishing  between  cystitis  and  pyelitis  by  the  examination 
of  the  urine.  The  chief  bacteria  causing  suppuration  in  the 
urinary  passages  are  : 

B.  Coli. — This  is  perhaps  the  commonest  form,  and  in  cystitis 
or  pyelitis  due  to  it  the  urine  remains  acid  unless  other  organisms 
gain  access. 

The  organism  can  usually  be  identified  with  a  fair  amount  of 
certainty  by  an  examination  of  an  unstained  hanging-drop  prepara- 
tion, when  numerous  short  bacilli  will  be  seen  in  active  movement, 
and  the  fluid  will  be  found  to  contain  pus  cells.  Follow  this 
examination  by  making  a  film  of  the  urine,  staining  by  Gram  and 
counterstaining  by  dilute  carbol  fuchsin,  when  the  bacilli  will  be 
seen  stained  red.  These  appearances  in  an  acid  urine  raise 
strong  presumptive  evidence  of  B.  coli,  but  are  not  conclusive,  as 
the  typhoid  bacillus  is  almost  identical.  Where  cocci  or  other 
organisms  are  present,  the  urine  may  be  alkaline  in  spite  of  the 
presence  of  B.  coli. 

Proteus  Vulgaris. — This  is  one  of  the  common  organisms  of 
suppuration,  and  produces  cystitis  with  an  alkaline  urine.  It 
often  occurs  in  conjunction  with  B.  coli,  and  the  urine  is  alkaline 
in  this  case  also,  the  proteus  being  more  powerful  as  a  producer 
of  alkali  (by  the  ammoniacal  decomposition  of  urea)  than  the 
B.  coli  is  of  acid. 

It  closely  resembles  B.  coli  in  appearance,  and  is  motile ;  the 
chief  points  of  difference  are  that  it  is  more  irregular  in  size  and 
forms  longer  threads,  and  that  some  of  the  bacilli  often  fail  to 
decolorize  by  Gram.  In  cases  where  the  two  organisms  are 
present  it  is  impossible  to  distinguish  the  one  from  the  other, 
either  in  a  hanging-drop  or  in  stained  films. 

If  it  is  necessary  to  know  whether  B.  coli  or  B.  typhosus  is 
present  along  with  Proteus  vulgaris  in  an  ammoniacal  urine,  the 
only    method   is    to    plate    out    some    of    the    urine  on   gelatin 


120  CLINICAL    BACTERIOLOGY   AND    HEMATOLOGY 

(see  p.  103),  making  several  plates  with  different  dilutions;  as  a 
rule,  a  minute  trace  of  the  urine  is  all  that  is  necessary.  Typhoid 
and  coli  form  small,  semi-translucent,  greyish  colonies  which  do 
not  tend  to  spread  very  much,  whilst  proteus  forms  small  grey 
colonies  which  form  radiating  branches  and  which  liquefy  the 
gelatin,  forming  a  saucer-like  excavation  with  a  little  white  mass 
of  growth  in  the  centre. 

Streptococci  occur  occasionally  as  independent  causes  of  pyelitis 
or  cystitis,  and  in  these  cases  I  believe  the  prognosis  to  be  some- 
what worse  than  in  the  other  forms. 

Staphylococci  usually  occur  as  a  secondary  infection  of  other 
forms  of  cystitis.  The  M.  tirece  is  a  frequent  cause  of  ammoniacal 
decomposition  of  the  urine,  and  is  often  mistaken  for  a  staphylo- 
coccus ;  the  two  are  indistinguishable  under  the  microscope, 
but  staphylococci  liquefy  gelatin  rapidly  and  M.  uvece  does  so 
slowly.  Their  separation  is  of  no  clinical  importance  in  these 
cases. 

The  M.  uvece  appears  to  be  identical  with  the  common  skin- 
coccus,  w^hich  is  the  most  common  and  characteristic  organism  of 
the  epidermis.  It  is  a  coccus  which  very  frequently  occurs  as  a 
contamination  in  cultures,  and,  as  a  consequence,  has  been 
accused  of  causing  an  abundance  of  diseases,  including  cancer. 

Gonococci. — When  these  are  found  in  the  urine  it  is  necessary 
to  consider  whether  they  come  from  the  bladder  or  urethra.  To 
do  so  it  usually  suffices  to  see  whether  they  occur  in  all  parts  of 
the  urine,  and  not  simply  in  the  first  portions,  which  wash  the 
pus  from  the  urethra. 

A  caution  is  necessary  in  the  diagnosis  of  gonorrhoeal  cystitis,  as 
some  cases  of  inflammation  of  the  bladder  (with  acid  urine)  are  due 
to  an  organism  closely  resembling  the  gonococcus,  but  a  little  larger 
and  more  variable  in  size  :  I  believe  it  to  be  M.  catarrhalis.  In 
cases  where  gonorrhoeal  cystitis  is  suspected  make  a  culture  on 
ordinary  agar  or  on  blood-serum.  The  diplococcus  in  question 
grows  readily,  the  gonococcus  does  not. 

Typhoid  Bacilli. — These  are  rare  causes  of  cystitis,  but  are 
commonly  found  in  the  urine  during  and  after  an  attack  of 
typhoid  fever.  When  they  produce  cystitis  the  urine  remains 
acid. 

They  cannot  be  distinguished  from  B.  coli  except  by  rather 
complicated  cultural  tests  or,  less  certainly,  by  means  of  the 
agglutination  reaction.     In  cases  where  they  are  suspected  the 


THE    URINE  121 

urine  must  be  plated  out  on  gelatin,  and  several  small  greyish, 
semi-transparent,  non-liquefying  colonies  picked  out  with  a  straight 
platinum  wire  and  each  transplanted  to  a  fresh  agar  tube.  After 
twelve  to  eighteen  hours'  incubation  there  should  be  a  delicate 
greyish  growth,  with  a  slight  tendency  to  spread  from  the  line  of 
inoculation.  This  is  to  be  tested  by  performing  Widal's  reaction 
with  it,  using  the  blood  from  a  patient  who  is  suffering  or  has 
suffered  from  typhoid  fever,  and  whose  blood  is  known  to  give  a 
Widal's  reaction  in  a  high  dilution— say,  i  in  200.  If  the  blood 
clumps  the  cultures  you  have  obtained  from  the  urine  when  used 
in  the  same  dilution,  the  organism  is  almost  certainly  the  typhoid 
bacillus. 

The  importance  of  this  examination  is  that  the  typhoid  bacillus 
may  be  excreted  in  the  urine  for  years  after  an  attack  of  typhoid 
fever,  causing  no  symptoms,  but  remaining  a  very  potent  cause  of 
infection.  Since  this  is  a  source  of  such  danger  to  others  it  is 
advisable  to  make  use  of  the  services  of  an  expert  (who  will  use 
better  methods  than  that  described)  in  the  examination  of  a 
suspected  case.  The  escape  of  the  bacilli  may  usually  be 
stopped  by  the  administration  of  urotropin. 

Bacteriuria,  or  Bacilluria,  is  a  term  which  should  be 
restricted  to  the  escape  of  bacteria  in  the  urine  without  the 
presence  of  pus  and  without  clinical  evidence  of  cystitis,  pyelitis, 
etc.  The  organisms  may  be  very  variable  ;  B.  coli  is  perhaps  the 
most  common. 

The  disease  may  be  diagnosed  when  a  urine  which  contains 
numerous  bacteria  is  found  on  several  occasions  to  be  free 
from  pus. 

THE  COLLECTION  OF  FLUIDS  FROM  SEROUS 
CAVITIES 

A  bacteriological  examination  of  the  inflammatory  exudates 
which  collect  in  the  various  cavities  of  the  body  often  yields 
important  information  as  to  the  nature  of  the  morbid  process, 
suggests  treatment,  and  influences  our  views  as  to  the  prognosis 
of  the  condition.  This  is  especially  the  case  with  the  fluids  which 
collect  in  the  pleura,  the  membranes  of  the  brain  and  cord,  and 
the  joints.  In  some  cases  all  the  needful  information  may  be 
obtained  by  the  examination  of  stained  films,  cultures  being  un- 
necessary ;  and  in  these  cases  no  antiseptic  or  aseptic  precautions 


122  CLINICAL    BACTERIOLOGY    AND    HEMATOLOGY 

(other  than  those  which  are  dictated  by  the  interests  of  the 
patient)  are  necessary.  But  in  the  greater  proportion  of  cases 
this  is  not  enough,  and  cultures  must  be  obtained.  To  this  end 
it  is  absolutely  essential  that  the  most  scrupulous  precautions  should 
be  taken  against  contamination  of  the  fluid  by  the  organisms 
which  are  constantly  present  in  the  air  and  in  the  skin,  or  the 
results  will  be  worthless.  The  precautions  taken  must  be  as 
complete  as  those  which  are  used  before  an  operation  upon  a 
joint.  Indeed,  a  fresh  precaution  has  to  be  taken,  for  whereas 
the  presence  in  the  skin  of  a  small  quantity  of  an  antiseptic 
would  not  be  detrimental  to  a  surgical  operation,  it  might,  by 
getting  into  the  fluid,  nullify  a  bacteriological  examination. 
Hence  the  skin  must  be  aseptic,  and  free  from  any  antiseptic 
chemical. 

The  technique,  as  far  as  aseptic  precautions  are  concerned,  is 
as  follows:  The  skin  at  the  region  to  be  punctured  is  first 
thoroughly  cleaned  with  soap,  hot  water,  and  (if  the  patient  can 
stand  it)  a  nail-brush.  Then  layer  after  layer  of  some  reliable 
antiseptic  lotion  is  painted  on,  each  layer  being  allowed  to  soak 
in  before  the  next  is  applied.  The  most  suitable  lotions  for  the 
purpose  are  perchloride  of  mercury  (i  in  i,ooo),  biniodide 
of  mercury  (i  in  500  of  methylated  spirit),  or  carbolic  acid 
(i  in  20). 

After  being  allowed  to  act  for  at  least  ten  minutes,  the  skin  is 
to  be  thoroughly  cleansed  with  methylated  spirit ;  this  should  be 
rubbed  in  with  a  piece  of  cotton -wool,  and  should  be  poured 
copiously  over  the  area.     The  operation  may  now  proceed. 

Where  possible  it  is  preferable  to  apply  a  dressing  of  lint 
soaked  in  one  of  the  above  lotions  (which  need  be  of  only  half 
the  strength)  for  a  few  hours. 

The  puncture  may  be  made  by  using  some  sort  of  exploring 
syringe,  or  a  hollow  needle  without  any  means  for  aspiration. 
The  former  is  used  for  the  removal  of  fluid  from  the  pleura 
or  synovial  cavity,  the  latter  in  performing  lumbar  puncture. 
But  it  is  necessary  that  the  whole  of  the  instrument  used  should 
be  rendered  sterile  by  heat;  chemical  antiseptics  are  as  a  rule 
inadmissible.  In  the  case  of  a  hollow  needle  no  difficulty  occurs  ; 
some  hypodermic  syringes,  however,  will  not  stand  boiling,  and 
these  are  useless  for  the  purpose,  as  the  apparatus  which  is  to 
be  used  must  be  boiled  for  at  least  five  minutes. 

We  shall  now  deal  with  the  most  important  cavities  of  the 


THE    PLEURA  123 

body,  describing  the  methods  to  be  employed  in  the  investigation 
of  the  inflammatory  exudates  which  they  may  contain,  and  the  infer- 
ences which  may  be  drawn  from  the  results  of  the  examination. 

THE  PLEURA 

There  is  but  little  to  be  said  about  the  method  to  be  employed 
in  the  collection  of  fluid  from  the  pleural  cavities.  The  most 
careful  antiseptic  precautions  are  to  be  taken,  and  the  region  to  be 
punctured  should  be  decided  by  consideration  of  the  physical  signs. 

The  examination  of  the  fluid  thus  obtained  may  be  either 
microscopical,  cultural,  or  by  injections  into  animals.  If  the 
latter  are  required  (and  inoculation  should  be  performed  in  all 
cases  where  a  tuberculous  origin  is  suspected)  a  considerable 
quantity  of  the  fluid — an  ounce  or  more — should  be  enclosed  in 
a  bottle  which  has  been  sterilized  by  boihng,  and  forwarded  at 
once.  A  drachm  or  more  of  lo  per  cent,  sodium  citrate  solution 
(boiled)  should  be  added  to  prevent  coagulation. 

Where  the  diagnosis  is  to  be  made  by  cultural  methods,  and 
the  cultures  are  not  to  be  made  on  the  spot,  the  fluid  is  best 
stored  or  sent  to  a  laboratory  in  pipettes.  These  are  to  be  filled 
from  the  syringe  direct ;  the  needle  is  to  be  removed,  and  the  end 
of  the  pipette  (sterilized  by  being  passed  through  the  flame)  is 
passed  into  the  fluid,  and  filled  by  gentle  suction  at  the  other  end. 
Each  end  is  then  sealed  in  a  flame,  care  being  taken  not  to  heat 
the  fluid.     Two  or  three  such  tubes  should  be  sent. 

Clear  fluid  from  the  chest  rarely  shows  any  micro-organisms  on 
microscopical  examination.  Cultures  are  usually  sterile  ;  where 
streptococci  or  pneumococci  are  found  the  inflammation  is  likely 
to  pass  on  into  suppuration.  The  great  majority  of  these  cases 
of  "simple  "  acute  pleurisy  are  really  due  to  the  tubercle  bacillus, 
but  their  true  nature  is  difiicult  to  determine  except  by  inoculation 
experiments.  A  cytological  examination  of  the  fluid  should  be 
made,  and  in  tuberculous  cases  the  cells  present  will  usually  be 
found  to  be  lymphocytes,  with  an  occasional  admixture  of  blood- 
corpuscles  (see  p.  237).  If  the  fluid  does  not  clot  spontaneously,  it 
may  be  very  thoroughly  centrifugalized,  and  films  prepared  from 
the  deposit  and  stained  for  tubercle  bacilli  in  the  ordinary  way  ; 
but  they  are  not  always  found  even  in  true  cases  of  tuberculous 
pleurisy.  Where  the  fluid  coagulates  spontaneously,  the  best 
plan  is  to  allow  the  clot  to  retract  until  it  has  shrunk  to  a  small 


124  CLINICAL    BACTERIOLOGY   AND    HEMATOLOGY 

volume,  to  remove  it  from  the  rest  of  the  fluid,  allowing  all  that  will 
to  drain  away,  and  then  to  digest  the  fibrinous  mass  in  an  artificial 
digestion  mixture  (pepsin  and  o*2  per  cent.  HCl)  until  completely 
dissolved.  The  resulting  fluid  is  now  centrifugalized  or  allowed 
to  stand  for  a  day  or  so  (in  which  case  some  thymol  should  be 
added  to  prevent  excessive  growth  of  bacteria),  and  films  prepared 
from  the  deposit,  which  will  contain  the  tubercle  bacilli.  The 
advantage  of  this  method  is  that  all  the  bacilli  in  four  or  five 
ounces  of  pleuritic  fluid  may  be  entangled  in  the  clot,  and  con- 
centrated into  a  comparatively  small  bulk.  The  tubercle  bacilli 
resist  peptic  digestion  for  a  long  time,  but  other  organisms  do 
not,  and  the  method  is  not  available  for  them. 

Purulent  pleurisies  (empyemata)  may  be  caused  by  many 
organisms,  the  most  common  being  the  pneumococcus,  strepto- 
cocci, staphylococci,  and  the  tubercle  bacillus. 

The  pneumococcus  is  readily  demonstrated  by  a  microscopical 
examination,  the  method  to  be  employed  being  the  same  as  that 
previously  described. 

The  pus  in  these  cases  is  thick  and  creamy,  and  of  a  greenish 
colour  ;  after  it  has  stood  for  some  time  a  thin  layer  of  a  greenish 
fluid  appears  upon  the  surface. 

When  an  empyema  is  due  to  the  pneumococcus  alone,  no  other 
organisms  being  present,  the  prognosis  is  distinctly  better  than  in 
cases  in  which  other  organisms  are  present,  and  the  patient  often 
recovers  "after  simple  aspiration.  This  is  especially  the  case  in 
children,  in  whom  empyema  is  due  to  this  organism  in  a  very 
large  number  of  cases,  certainly  over  90  per  cent. 

If  cultures  are  made  in  pneumococcic  cases,  it  may  be  noted 
that  occasionally  very  few  of  the  organisms  appear  to  grow  into 
colonies,  as  far  as  can  be  judged  by  a  comparison  between  the 
numbers  of  cocci  present  in  the  films  and  of  colonies  on  the  tubes. 
This  indicates  that  the  majority  of  cocci  are  dead,  and  this  makes 
the  prognosis  better.  The  prognosis  is  also  good  in  cases  in  which 
very  few  cocci  are  present,  and  in  those  in  which  the  cocci  that 
are  present  are  largely  contained  in  the  leucocytes  ;  in  this  case 
they  may  lose  their  power  of  retaining  Gram's  stain. 

The  streptococcus  is  also  readily  demonstrated  by  a  simple  micro- 
scopical examination ;  it  grows  readily  on  agar,  forming  small 
round  colonies,  which  do  not  tend  to  coalesce  and  are  more  opaque 
in  the  centre  than  at  the  periphery. 

The  pus  is  not  generally  very  thick,  and  has  a  yellow  colour 


THE    PLEURA  I25 

It  separates  into  two  layers,  the  upper  transparent  layer  being 
much  more  abundant  than  is  the  case  with  pneumococcic  pus. 

This  form  of  empyema  is  rare  in  children,  but  is  perhaps,  on 
the  whole,  the  commonest  one  in  adults.     The  prognosis  is  much_ 
worse  than  in  the  pneumococcic  cases,  and  thorough  drainage  and 
resection  of  the  ribs  is  essential. 

Staphylococcic  empyemata,  according  to  Netter,  are  very  rare  ; 
the  single  case  in  which  he  found  the  staphylococcus  alone  was 
secondary  to  ulcerative  endocarditis.  He  also  states  that  when 
this  organism  is  found  in  the  pus  tubercle  bacilli  are  often  present  as 
well.     The  prognosis  of  these  cases,  therefore,  appears  to  be  bad. 

The  Uihevcle  bacillus  is  responsible  for  a  comparatively  small 
number  of  cases,  and  the  results  of  operative  interference  are 
not  gratifying.  The  prognosis  is  worse  than  in  any  other  form  of 
the  disease. 

The  pus  is  usually  white  in  colour^  and  thin  and  watery.  It 
may  contain  small  masses  of  white  caseous  material.  The  leuco- 
cytes which  it  contains  are  nearly  all  lymphocytes,  unless  a 
secondary  infection  with  pus  organisms  has  taken  place. 

The  diagnosis  may  be  made  from  a  careful  microscopical 
examination,  but  to  this  end  it  must  he  careful^  as  the  bacilli  are 
present  in  but  scanty  numbers. 

If  no  organisms  are  found  after  a  thorough  microscopical  ex- 
amination, the  inference  is  that  the  case  is  tuberculous.  If  a 
cultural  examination  is  also  negative  the  inference  becomes 
almost  a  certainty. 

The  empyemata  arising  from  rupture  of  the  oesophagus,  stomach, 
intestine,  etc.,  into  the  pleura,  in  those  due  to  an  external  wound 
with  free  contamination  of  the  membrane  and  its  contents,  and  in 
those  due  to  the  rupture  of  very  foul  tuberculous  vomicae,  contain 
vast  numbers  of  organisms  of  all  kinds — bacilli,  cocci,  etc. — mixed 
together.  These  fluids  usually  smell  badly,  and  are  of  very  evil 
omen. 

Lastly,  in  a  few  cases  other  organisms,  such  as  the  typhoid 
bacillus,  may  be  found. 

Having  these  facts  in  view,  the  practitioner  is  recommended  to 
proceed  to  examine  cases  of  purulent  pleurisy  in  the  following 
manner :  The  pus  is  to  be  withdrawn  with  a  hypodermic  needle 
or  exploring  syringe,  and  a  few  drops  deposited  at  once  on  the 
surface  of  a  culture-tube  of  agar,  spread  well  over  the  surface  with 
a  platinum  loop,  and  incubated  at  the  temperature  of  the  body. 


126  CLINICAL    BACTERIOLOGY   AND    HEMATOLOGY 

The  microscopical  examination  is  made  in  the  manner  described 
for  pus,  a  simple  stain  and  also  Gram's  stain,  with  dilute  carbol 
fuchsin  as  a  counterstain,  being  used.  The  presence  of  strepto- 
cocci, staphylococci,  and  pneumococci  will  be  revealed  ;  bacilli 
may  be  present,  and  in  this  case  it  should  not  be  forgotten  that 
the  tubercle  bacillus  stains  by  Gram's  method.  If  no  organisms  are 
found  in  these  films,  or  if  there  are  organisms  which  resemble  the 
tubercle  bacillus  in  general  appearance,  another  specimen  should 
be  submitted  to  prolonged  staining  in  hot  carbol  fuchsin  and  de- 
colorization  in  dilute  sulphuric  acid,  and  thoroughly  searched  for 
the  tubercle  bacillus.  If  the  result  is  negative,  several  other  films 
should  be  searched. 

The  cultures  are  to  be  examined  after  twenty-four  hours'  incu- 
bation. The  pneumococcus  will  produce  tiny  colourless  colonies 
on  the  surface  of  the  agar  ;  the  streptococcus  forms  similar  small 
colourless  colonies,  but  these  are  distinctly  more  opaque  in  the 
centre ;  staphylococci  form  opaque  white  or  yellowish  colonies 
which,  after  longer  incubation,  spread  out,  coalesce,  and  cover  the 
surface  of  the  agar  with  an  even  film  like  a  streak  of  paint ;  and 
the  tubercle  bacillus  does  not  develop.  Films  should  be  made 
from  the  cultures,  stained  and  examined.  The  cultural  examina- 
tion is  of  great  value,  but  much  can  be  made  out  by  the  examination 
of  stained  films  made  directly  from  the  pus. 

FLUIDS  FROM  JOINTS 

The  technique  of  the  process  of  withdrawing  these  fluids  is 
exactly  the  same  as  in  the  case  of  pleurisy ;  the  needle  will 
naturally  be  inserted  at  a  point  where  there  is  definite  evidence  of 
the  presence  of  fluid,  and  where  it  lies  near  the  surface. 

The  bacteriological  examination  is  conducted  on  exactly  similar 
lines.  A  few  drops  of  the  fluid  should  be  allowed  to  flow  on  to 
the  surface  of  a  sloped  tube  of  agar,  and  the  culture  obtained 
after  twenty-four  hours'  incubation  examined  in  the  manner 
already  described.  Films  should  also  be  made  directly  from  the 
fluid,  and  some  stained  by  Gram's  method  and  others  by  a  simple 
stain  such  as  carbol  thionin. 

A  great  number  of  organisms  may  be  present :  the  streptococci, 
staphylococci,  the  pneumococcus,  gonococcus,  and  tubercle  bacillus, 
are  the  most  important.  The  coccus  which  has  been  described 
by  several  observers  as  the  cause  of  acute  rheumatism  cannot  be 
considered  as  of  diagnostic  importance  at  present ;  the  same  remarl^ 


FLUIDS    FROM   JOINTS  I27 

applies  to  the  bacillus  which  is  possibly  the  cause  of  rheumatoid 
arthritis. 

Streptococci  are  readily  distinguished  on  microscopical  examina- 
tion, and  may  be  present  even  if  the  fluid  is  perfectly  clear.  When 
they  are  present  in  a  joint  which  is  not  the  seat  of  a  perforating 
wound,  they  indicate  a  general  infection  with  the  streptococcus, 
ulcerative  endocarditis,  etc.,  and  the  prognosis  is  most  grave.  The 
author  was  enabled  to  diagnose  a  case  of  streptococcic  septicaemia 
a  few  hours  after  the  onset  of  symptoms  by  finding  numerous 
chains  in  a  single  drop  of  clear  fluid  aspirated  from  the  knee-joint. 
The  clinical  aspect  was  at  that  time  very  similar  to  that  of  severe 
rheumatism,  and  the  case  had  been  so  diagnosed. 

In  such  cases  the  use  of  antistreptococcic  serum  offers  some 
hope  to  the  patient,  and  should  be  tried. 

Staphylococci  are  generally  found  in  cases  of  arthritis  due  to 
perforating  wounds,  or  in  the  course  of  a  general  infection.  They 
may  also  occur  along  with  the  gonococcus  in  cases  of  gonorrhoeal 
arthritis. 

The  pneumococcus  occurs  in  general  infection  from  a  primary 
focus  in  the  lung,  middle  ear,  etc.,  or  in  the  course  of  ulcerative 
endocarditis,  and  may  also  occur  as  a  primary  infection — at  least, 
in  cases  in  which  no  other  lesion  is  found.  The  prognosis  appears 
on  the  whole  to  be  fairly  good  in  these  cases  of  suppurative 
arthritis  of  pneumococcic  origin,  and  complete  recovery  with  a 
fully  movable  joint  may  occur. 

The  gonococcus  occurs  in  some  cases  of  gonorrhoeal  arthritis ;  it 
may  be  present  in  pure  culture,  or  it  may  be  mixed  with  other 
organisms,  especially  the  pus  cocci.  In  other  cases  of  gonorrhoeal 
arthritis  no  bacteria  are  found,  either  microscopically  or  on  cultural 
examination,  and  in  these  the  bacteria  have  probably  died  out 
before  the  fluid  was  withdrawn  or  are  localized  deep  down  in  the 
tissues. 

The  tubercle  bacilhis  may  be  found  in  cases  of  tuberculous  syno- 
vitis, but  it  is  more  probable  that  the  most  careful  search  will  be 
unsuccessful.  If  bacilli  having  the  general  appearance  of  this 
organism  are  found  in  the  Gram  specimen,  the  carbol  fuchsin 
method  of  staining  should  be  applied  to  a  fresh  film. 

Fluid  from  a  joint  may  be  sterile  in  cases  of  tubercular  synovitis, 
gonorrhoeal  arthritis,  synovitis  due  to  an  aseptic  injury, rheumatism, 
gout,  or  in  rheumatoid  arthritis,  etc. 


128  CLINICAL   BACTERIOLOGY   AND    HEMATOLOGY 


LUMBAR  PUNCTURE 

Fluid  may  be  removed  from  the  spinal  meninges  for  a  bacterio- 
logical or  other  examination  by  means  of  Quincke's  lumbar 
puncture.  The  information  furnished  by  this  means  is  often  of 
very  great  value ;  in  fact,  Osier  says  that  *'  during  the  past  ten 
years  no  single  measure  of  greater  value  in  diagnosis  has  been 
introduced."  The  process  is  simple,  easy,  and  entirely  devoid  of 
danger,  and  can  be  carried  out  without  an  anaesthetic.  I  have 
now  performed  this  operation  over  three  hundred  times,  and  have 
only  seen  bad  after-effects  (in  all  cases  temporary)  in  three  or  four 
cases,  in  all  of  which  eucaine  had  been  used  as  a  local  anaesthetic. 
I  have  therefore  abandoned  the  use  of  this  drug,  and  now  employ 
only  ethyl  chloride  locally.  On  the  other  hand,  a  very  large 
amount  of  benefit  is  often  derived  from  it,  and  it  must  be  regarded 
as  the  most  useful  therapeutic  agent  at  our  disposal  in  the  treat- 
ment of  meningitis,  especially  chronic  forms ;  one  case  at  least  of 
tuberculous  meningitis  has  been  cured  by  repeated  punctures. 
It  acts,  of  course,  by  relieving  the  high  intracerebral  tension  and 
by  removing  some  of  the  toxin-containing  cerebro-spinal  fluid. 
In  basic  meningitis  I  have  seen  several  cases  in  which  the 
patient's  life  was  saved  by  it :  the  urgent  pressure  symptoms 
were  relieved  on  several  occasions,  the  relief  lasting  a  few  days, 
and  he  was  thus  enabled  to  tide  over  the  attack,  uninterrupted 
and  complete  recovery  resulting.  In  a  case  in  which  I  performed 
the  operation  recently  a  single  puncture  was  followed  by  imme- 
diate relief,  though  the  symptoms  were  most  grave,  and  there  has 
been  at  present  (after  a  month)  no  recrudescence.  Great  temporary 
relief  may  also  be  given  in  the  severe  headache  of  uraemia  and 
chlorosis. 

It  should  never  be  done  on  a  "  walking  "  patient,  since  if  too 
much  fluid  is  drawn  off  headache  or  vomiting  are  said  to  result ; 
these  soon  pass  off  if  the  patient  rests  in  bed. 

Requisites. — i.  A  suitable  needle.  In  children  the  spinal 
meninges  will  be  reached  at  a  depth  of  3  to  4  centimetres  (roughly 
I  to  i^  inches),  whilst  in  adults  the  depth  may  be  twice  as  great. 
The  needle  should  not  be  less  than  2I  inches  long  for  an  infant 
and  4  inches  for  an  adult,  and  should  be  sharp  and  strong.  An 
antitoxin  needle  will  usually  serve  very  well.  A  syringe  is 
.unnecessary,   and  the  fluid  should  never  be  sucked  out  of  the 


LUMBAR    PUNCTURE  •  I29 

spine,  or  injury  to  the  nerve-roots  may  result.  The  needle  should 
always  have  a  sharp  and  stiff  wire  inside  it  when  the  puncture  is 
made,  otherwise  it  may  become  blocked  by  a  shred  of  fibrous 
tissues  picked  up  during  its  passage  through  the  parietes,  and 
time  may  be  lost  in  sterilizing  a  wire  with  which  to  clear  it. 
Another  use  for  the  wire  arises  from  the  fact  that  when  the 
intracerebral  pressure  is  low  the  needle  may  push  the  membrane 
in  front  of  it,  instead  of  perforating  it :  when  this  is  the  case  a 
short  sharp  push  with  the  wire  will  probably  puncture  the 
membrane,  and  the  point  of  the  needle  will  slip  in.  A  piece  of 
steel  wire  is  best. 

Since  cultures  may  have  to  be  taken,  it  is  advisable  to  have 
some  sort  of  a  handle  which  may  be  sterilized  with  the  needle  and 
avoid  any  necessity  for  a  very  elaborate  sterilization  of  the  hands. 
I  use  a  pair  of  artery  forceps,  one  blade  of  which  is  passed  inside 
the  barrel  and  the  other  outside,  and  the  instrument  clipped  on. 


Fig.  30. — Author's  Needle  for  Lumbar  Puncture. 

The  two  are  sterilized  together  and  the  forceps  used  as  a  handle, 
and  not  removed  until  the  puncture  has  been  made.  Where 
a  sterilizer  large  enough  to  take  the  two  instruments  fixed 
together  is  not  at  hand  the  needle  may  be  placed  point  down- 
ward in  a  test-tube  half  full  of  water,  and  the  forceps  inserted 
as  far  as  it  will  go  by  its  side.  The  tube  is  boiled  for  ten 
minutes  and  the  needle  removed  by  clipping  it  with  the  forceps, 
as  above.  The  handle  will  not  be  sterilized,  but  that  does  not 
matter. 

I  have  devised  a  curved  needle  with  a  handle  fitting  into  a 
socket  (Fig.  30),  which  also  answers  well.  It  has  the  advantage 
that  it  can  be  put  in  a  wide  tube  plugged  with  cotton-wool, 
sterilized  by  dry  heat,  and  taken  to  the  bedside  ready  for 
immediate  use. 

2.  Materials  for  disinfection  of  the  patient's  skin  and  (if  cultures 
are  to  be  taken)  the  hands  of  the  operator.  Hot  water,  soap, 
alcohol,  ether,  perchloride  lotion  (i  in  1,000). 

9 


130  CLINICAL    BACTERIOLOGY   AND    H.^MATOLOGY 

3.  Apparatus  for  boiling  the  needle  in  a  dilute  solution  of 
washing  soda.* 

4.  Spray  for  local  anaesthesia.  If  this  is  used  the  hard  plaque 
of  skin  adds  considerably  to  the  difficulty  of  the  operation.  If, 
however,  the  region  be  frozen  twice,  and  allowed  to  thaw  after 
each  freezing,  the  skin  will  be  found  to  have  resumed  its  normal 
texture  and  to  be  very  fairly  anaesthetic. 

5.  Two  or  three  test-tubes  sterilized  by  dry  heat  and  plugged 
with  dry  cotton-wool. 

6.  If  cultures  are  to  be  taken,  the  tubes  of  medium  should  be 
inoculated  at  the  time  of  the  operation  if  possible.  The  medium 
required  will  depend  to  a  great  extent  upon  the  nature  of  the 
organism  which  is  expected.  If  there  are  no  indications  upon 
this  point,  the  most  suitable  is  solidified  blood-serum,  but  in 
default  of  this  ordinary  agar  will  answer  well.  If  the  case  is 
thought  to  be  one  of  cerebro-spinal  fever,  the  most  suitable 
medium  for  the  cultivation  of  the  specific  organism  (Weichsel- 
l)aum's  Diplococcus  intvacellularis)  is  alkaline  5  per  cent,  glycerin- 
agar,  and  a  couple  of  tubes  of  this  should  be  at  hand,  as  well  as 
blood-serum  or  ordinary  agar. 

Process — i.  Preliminary. — As  in  removal  of  fluids  for  bacterio- 
logical examination  from  other  parts  of  the  body,  it  is  better  if  the 
skin  can  be  sterilized  some  hours  before  the  operation,  and  a  pad 
soaked  in  an  antiseptic  fluid  kept  on  the  area  until  the  last 
moment.  This  is  usually  impracticable,  and  the  process  will  be 
described  as  if  it  were  performed  at  a  single  visit. 

Put  the  needle  to  boil  in  a  weak  solution  of  washing-soda,  and 
proceed  to  the  disinfection  of  the  patient's  back.  When  the 
needle  has  boiled  for  five  minutes,  remove  the  vessel  from  the 
flame  and  allow  it  to  cool  without  removing  the  needle. 

Place  the  patient  on  his  left  side,  and  find  the  processes  of  the 
second,  third,  and  fourth  lumbar  vertebrae.  A  line  drawn  between 
the  upper  points  of  the  iliac  crests  usually  cuts  the  spine  at  the 
upper  edge  of  the  spinous  process  of  the  fourth  lumbar  vertebra. 
Scrub  the  skin  in  this  vicinity  with  soap  and  hot  water  ;  wash  the 
region  with  alcohol  and  then  with  ether,  and  allow  it  to  dry  ;  paint 
on  several  layers  of  perchloride  lotion,  allowing  each  to  soak  in 
before  the  next  is  applied ;  cover  the  region  with  a  piece  of  lint 

*  If  possible  the  needle  should  be  sterilized  by  dry  heat  previous  to  the 
operation,  and  kept  in  a  tube  plugged  at  both  ends  with  cotton-wool,  as  in  the 
method  recommended  for  the  collection  of  blood  for  bacteriological  examination. 


I 


LUMBAR    PUNCTURE  ,        I3I 

soaked  with  lotion,  and  proceed  to  disinfect  your  hands.  Lastly, 
pour  some  alcohol  on  to  the  skin  of  the  patient's  back  to  wash  off 
the  excess  of  the  antiseptic. 

2.  Operation — Position. — Get  the  patient  (still  lying  on  his  left" 
side)  to  draw  up  his  knees  so  as  to  flex  his  back  somewhat,  and  to 
turn  partly  over  on  to  his  face. 

Identify  the  processes  of  the  third  and  fourth  lumbar  vertebrae, 
and  mark  the  centre  of  the  space  between  them  by  means  of  the 
index-finger  or  thumb  of  the  left  hand.  If  local  anaesthesia  is  to 
be  employed  freeze  the  skin  round  a  point  about  J  inch  to  the 
right   of  the   middle   line,    opposite   the   spot    marked    by  your 


Fig.  31. — Operation  of  Lumbar  Puncture  with  Patient  in 
Sitting  Position. 

left  finger  or  thumb.  Take  the  needle  in  the  right  hand,  holding 
it  like  a  pen,  and  enter  it  at  a  point  level  with  the  centre  of  the 
interspace,  and  i  centimetre  (a  little  less  than  J  inch)  to  the 
right  of  the  middle  line.  Direct  it  forwards,  slightly  upwards,  and 
slightly  inwards,  and  press  it  in  with  a  steady  and  uniform  pressure ; 
this  must  be  applied  accurately  in  the  axis  of  the  needle,  or  the 
latter  may  bend  and  take  a  wrong  direction. 

If  the  needle  strikes  against  bone  withdraw  it  almost  completely, 
and  push  it  on  again  after  changing  its  direction  slightly.  If  bone 
is  again  encountered  it  may  be  advisable  to  try  again  in  the  inter- 
space between  the  second  and  third  processes. 

9—2 


132        .   CLINICAL   BACTERIOLOGY   AND    H.53MATOLOGY 

If  the  patient  is  able  to  do  so  without  harm,  it  is  much  easier 
to  perform  the  operation  with  him  sitting  in  a  chair  leaning  over 
the  back  or  on  the  edge  of  the  bed  with  his  head  nearly  between 
his  knees,  so  that  the  spine  is  bent  into  a  convex  curve ;  by  this 
means  the  spinous  processes  are  rendered  more  prominent  and 
the  spaces  between  the  laminae  are  made  wider  (see  Fig.  31).  The 
operation  is  thus  made  much  more  easy,  and  the  method  is  usually 
preferable  in  cases  of  tabes  or  general  paralysis,  where  the  fluid 
is  collected  for  a  cytological  examination  only,  and  the  patient  is 
in  good  general  health.  But  the  method  with  the  patient  on  his 
side  should  also  be  learnt. 

3.  Collection  of  Fluid  and  Inoculation  of  Media. — The  first  few 
drops  of  fluid  which  escape  may  be  stained  with  blood  ;  in  this 
case  they  should  be  rejected.  Allow  a  few  drops  of  the  fluid  to 
flow  directly  on  to  the  surface  of  the  medium  without  touching  the 
glass.  Collect  also  some  of  the  fluid  (i  to  2  drachms)  in  the 
sterilized  empty  tube.  If  no  fluid  flows  through  the  needle,  it  is 
presumptive  evidence  against  the  presence  of  acute  meningitis.  A 
*'  dry  tap"  may,  however,  occur  from  plugging  of  the  needle  with 
fibrin,  or  from  its  point  coming  in  contact  with  a  nerve  root 
(Osier),  and  in  some  cases  of  meningitis  the  purulent  exudation  is 
too  thick  to  flow  through  the  needle.  ,  In  one  case  in  which  no 
fluid  could  be  obtained  by  repeated  punctures  the  venous  sinuses 
of  the  brain  were  found  to  be  thrombosed  :  a  frequent  cause  in 
meningitis  is  the  closure  of  the  foramina  in  the  roof  of  the  fourth 
ventricle. 

The  force  of  the  flow  should  be  noted.  In  health  it  flows 
out  slowly,  whilst  in  meningitis  it  runs  faster,  and  may  even  spurt 
out  a  foot  or  more;  the  same  thing  may  happen  if  there  is  a 
cerebral  tumour,  uraemia,  or  other  cause  of  increased  pressure. 

4.  Examination  of  the  Fluid. — {a)  Naked-Eye. — When  meningitis 
is  present  the  fluid  is  always  more  or  less  turbid,  and  some 
observers  hold  that  the  turbidity  is  greater  in  proportion  to  the 
severity  of  the  case.  Osier  has  pointed  out  that  the  fluid  may  be 
alternately  turbid  and  clear,  being  clear  during  the  remissions  and 
turbid  during  the  exacerbations  of  the  disease.  Blood-stained 
fluid  may  occur  in  meningitis  or  from  haemorrhage  into  the 
cerebral  or  spinal  meninges  apart  from  inflammation."*  The 
presence  of  clear  fluid  aflbrds  strong  evidence  of  the  absence  of 
meningitis,  but  in  tuberculous  meningitis  the  amount  of  turbidity 

*  See  also  p.  242. 


LUMBAR   PUNCTURE  I33 

may  be  very  slight.  It  should  be  estimated  by  comparing  the 
fluid  with  some  distilled  water  in  a  clean  test-tube  of  the  same 
size  in  the  two  cases. 

(b)  Microscopical  —Prepare  films  of  the  exudate  in  the  manner^ 
recommended  on  p.  135  if  the  fluid  is  thin  and  watery;  if  it  is 
thick  and  purulent,  treat  it  Hke  ordinary  pus.    Stain  by  any  of  the 
methods  recommended  for  the  examination  of  the  blood  (Jenner's 
stain  being  most  convenient),  and  examine.* 

The  presence  of  leucocytes  (except  in  very  small  numbers) 
indicates  meningitis.  If  the  bulk  of  the  leucocytes  are  lympho- 
cytes (indicated  by  their  small  size,  large,  circular,  deeply  staining 
nuclei,  and  absence  of  granules)  the  presumption  is  that  the  case 
is  one  of  tuberculous  meningitis.  In  acute  meningitis  due  to  other 
bacteria  the  chief  cell  is  the  polynuclear  leucocyte  ;  this  may  be 
recognised  by  its  larger  size,  its  twisted  (apparently  multiple) 
nucleus,  and,  if  the  staining  method  has  been  appropriate,  by  the 
presence  in  its  protoplasm  of  minute  granules  which  stain  with  eosin. 
The  fluid  may  also  contain  red  blood-corpuscles  and  shreds  of  fibrin. 

{c)  Chemical. — Cerebro- spinal  fluid  removed  from  a  person  who 
is  not  suffering  from  meningitis  contains  a  very  minute  amount 
of  albumin,  while  when  the  meninges  are  inflamed  the  quantity 
is  greatly  increased.  The  method  of  testing  these  small  amounts  of 
albumin  quantitatively  is  hardly  within  the  reach  of  practitioners; 
if  a  considerable  amount  of  fluid  has  been  obtained,  a  small 
quantity  should  be  tested  by  heat  and  acetic  acid,  and  the  amount 
of  opacity  noted.  This  should  be  very  slight,  not  more  than  what 
would  be  called  a  "  faint  haze  "  in  urinary  work. 

Another  test  which  has  almost  escaped  notice,  but  which  I 
regard  as  one  of  the  most  important,  is  that  for  the  presence  of 
sugar.  For  this  (as  for  the  testing  for  albumin)  the  clear  super- 
natant fluid  left  after  centrifugalization  should  be  used.  In  health, 
sugar  is  present  in  considerable  amount  (about  0*06  per  cent.),  and 
Fehling's  solution  is  vigorously  reduced  ;  in  meningitis,  sugar  is 
either  reduced  to  a  trace  or,  much  more  frequently,  completely 
absent.  I  have  only  found  this  test  fail  once  in  about  a  hundred 
cases  (even  in  this  one  the  diagnosis  was  never  made  absolutely 
certain),  and  regard  it  as  the  most  certain  single  sign  of  meningitis.! 

*  The  cytology  of  the  cerebro-spinal  fluid  is  dealt  with  more  fully  under  the 
heading  of  Cyto-diagnosis. 

t  Since  writing  the  above  I  have  met  with  several  cases  of  tuberculous 
meningitis  in  which  sugar  was  present.  The  reduction  of  the  Fehling  is, 
however,  but  slight,  and  the  oxide  may  not  appear  for  several  minutes. 


134  CLINICAL   BACTERIOLOGY   AND    HEMATOLOGY 

[There  are  also  characteristic  chemical  changes  in  uraemia,  to 
which  I  have  paid  a  good  deal  of  attention  of  late.  In  health  the 
cerebro-spinal  fluid  contains  about  0*035  to  0-04  per  cent,  of  urea, 
07  per  cent,  of  chlorides,  and  has  a  freezing-point  of  —0-56°  C,  or 
thereabouts.  In  renal  disease  in  which  the  kidneys  are  acting 
well  these  figures  are  but  slightly  disturbed,  but  in  uraemia  there 
is  abundant  evidence  of  retention  of  soluble  substances.  The  urea 
increases  greatly — the  maximum  I  have  seen  being  0-4  per  cent. — 
the  chlorides  may  rise  to  i  per  cent.,  and  the  freezing-point  is 
depressed.  I  believe  this  to  be  the  simplest  test  of  the  functional 
capacity  of  the  kidney,  the  chemical  examinations  being  easy  in  a 
substance  of  such  simple  constitution  as  the  cerebro-spinal  fluid. 
It  is  occasionally  of  value  in  patients  found  unconscious.  In 
one  such  case  I  was  able  to  exclude  uraemia  definitely,  though 
the  urine  contained  albumin  and  casts  :  it  turned  out  to  be  a  case 
of  poisoning.  In  uraemia  there  is  often  the  additional  advantage 
of  its  affording  a  relief  to  some  of  the  symptoms.  Further  details 
are  outside  the  scope  of  this  work.] 

(d)  Bacteviological. — The  chief  organisms  which  cause  acute 
meningitis  are  given  in  the  following  table,  which  is  modified 
from  one  given  by  Osier  : 

Primary  {i.e.,  not  dependent  on  an  obvious  lesion  elsewhere  in  the  body). 

1.  Cerebro-spinal  fever— 

,,,^   . ,      .    IWeichselbaum's  diplococcus,  or  meningococcus. 
(6)  Epidemicj 

2.  Pneumococcic — 

{a)  Pneumococcic  infection  of  meninges  alone,  not  de-' 
pendent  on  disease  of  distant  parts  of  the  body. 

{b)  Pneumococcic  infection  of  meninges  occurring  as  |.Pneumococcus. 
part  of  a  general  septicaemia  without   obvious 
primary  lesion. 

Secondary. 

A.  To  direct  extension  from  local  disease  of  the  cranium,  middle  ear,  fossae, 

spinal  column,  etc. 
Pneumococcus. 
Staphylococci. 
Streptococci,  etc, 

B.  To  septicaemic  infection  due  to  disease  in  a  distant  part  of  the  body. 

{a)  Pneumococcic — 

Secondary  to  pneumonia,  endocarditis,  etc. 
Pneumococcus. 


LUMBAR   PUNCTURE  I35 

(b)  Pyogenic — 

Secondary  to  abscesses,  etc.,  and  occurring  as  a  part  of  a  general 
infection. 
Staphylococci. 
Streptococci,  etc.  _ 

(c)  Gonorrhoeal — 

Secondary  to  gonorrhoea. 
Gonococcus  (rare). 

(d)  Tuberculous- 

Secondary  to  tuberculosis  of  other  regions. 
Tubercle  bacillus. 

(e)  Miscellaneous — 

Secondary  to  typhoid  fever. 

Typhoid  bacillus  (rare). 
Secondary  to  influenza. 

Influenza  bacillus  (rare). 
Secondary  to  anthrax,  etc. 

Anthrax  bacillus,  etc.  (rare). 

Of  these,  the  organisms  which  are  most  likely  to  occur  are  : 

The  tubercle  bacillus  )  ,■      r       ^      .  .      e 

I  accounting  tor  about  go  per  cent,  of  cases. 
Meningococcus  > 

The  pneumococcus. 

Streptococci  and  staphylococci  in  secondary  cases  due  to  spread  from 

ear,  etc. 

And  this  shows  roughly  the  order  of  frequency. 

Preparation  of  Films. — If  the  fluid  is  thick  and  purulent,  films 
should  be  prepared,  dried,  and  fixed  in  the  ordinary  way.  If  the 
fluid  is  thin  and  watery,  it  should  be  centrifugalized  or  allowed 
to  stand  for  some  hours.  A  certain  amount  of  coagulation  will 
take  place,  and  the  sediment  which  collects  will  contain  the  bulk 
of  micro-organisms.  The  clear  fluid  should  be  poured  off  and 
used  for  testing  chemically,  and  the  sediment  used  for  the  pre- 
paration of  films,  of  which  several  will  be  required.  The  subse- 
quent examination  will  depend  to  some  extent  upon  the  nature  of 
the  organism  which  is  probably  present ;  for  general  purposes 
stain  one  or  more  films  with  Loffler's  methylene  blue  (two 
minutes),  wash,  dry,  mount,  and  examine. 

Streptococci  and  staphylococci  will  be  readily  recognised  by 
their  morphological  characters.  If  diplococci  are  present  they 
may  be  pneumococci,  Weichselbaum's  diplococci,  or  gonococci. 
Stain  a  film  by  Gram's  method  and  counterstain  in  dilute  carbol 
fuchsin  in  the  method  described  for  the  gonococcus.  Pneumococci 
will  retain  the  violet  stain,  while  Weichselbaum's  organism  and 
gonococci  will  be  coloured  red. 


136  CLINICAL    BACTERIOLOGY    AND    HEMATOLOGY 

Weichselbaum's  Diplococcus  meningitidis  intvacellulavis  is  now 
generally  considered  to  be  the  specific  cause  of  cerebro-spinal 
fever.  It  is  a  diplococcus  which  varies  very  considerably  in  size, 
but  is  usually  rather  smaller  than  the  gonococcus.  The  opposed 
surfaces  of  two  cocci  making  up  the  pair  are  usually  somewhat 
flattened,  but  this  is  not  so  marked  as  in  the  latter  organism.  It 
is  often  contained  within  the  polynuclear  leucocytes,  but  is  not 
grouped  in  large  numbers  in  a  single  cell — others  being  free — as 
is  usually  the  case  with  the  gonococcus  (see  Plate  III.,  where  the 
two  organisms  are  contrasted).  The  two  also  differ  in  their  cultural 
characters  and  in  their  pathogenicity  to  animals.  If  any  question 
should  arise  as  to  which  of  the  two  is  present  in  the  meningeal 
exudation  in  a  case  in  which  no  cultures  have  been  taken,  other 
evidence  of  gonorrhoeal  infection  should  be  sought  for. 

Still's  diplococcus  of  posterior  basic  meningitis  cannot  be 
distinguished  from  the  D.  meningitidis  by  its  morphological 
characters  alone,  and  many  bacteriologists  think  the  two 
organisms  are  in  reality  identical.  The  truth  seems  to  be  that 
the  organism  which  we  call  the  meningococcus  is  a  very  variable 
one,  some  forms  being  much  more  easily  cultivated  than  others, 
and  also  differing  in  a  few  other  minute  points.  Yet  these  forms 
appear  to  occur  indiscriminately  in  true  epidemic  cerebro-spinal 
meningitis  and  in  posterior  basic  meningitis,  and  these  diseases 
are  quite  indistinguishable  on  pathological  grounds. 

The  Rarer  Causes  of  Meningitis. — The  bacilli  of  typhoid  fever, 
anthrax,  influenza,  etc.,  may  also  be  recognised  in  the  methylene 
blue  specimen,  and  should  be  identified  (if  possible)  by  a  careful 
study  of  their  morphological  appearances  and  reaction  to  Gram's 
stain. 

If  no  organisms  are  found  in  the  methylene  blue  specimens 
after  a  careful  search,  and  if  the  characters  of  the  fluid  are  such 
as  indicate  that  meningitis  is  present,  the  presumption  is  that  the 
case  is  one  of  tuberculous  meningitis.  Films  should  be  stained 
in  the  method  already  described  and  carefully  searched ;  the 
bacilli  are  present  in  very  scanty  numbers,  and  many  films  may 
have  to  be  examined  before  one  is  found. "^ 

*  Lenharz  adds  a  shred  of  clean  cotton-wool  to  the  fluid.  This  sinks  slowly 
to  the  bottom,  and  is  withdrawn  after  some  hours,  spread  on  a  slide,  dried, 
and  stained  for  tubercle  bacilli.  The  author  has  had  no  experience  of  this 
method,  but  Mr.  Leedham-Green  informs  him  that  it  is  of  considerable  value. 
(See  also  p.  138). 


LUMBAR   PUNCTURE  I37 

Cultural  Examination. — The  tubes  which  have  been  inoculated 
by  allowing  the  fluid  to  drop  directly  on  to  the  surface  of  the 
medium  are  to  be  incubated  for  twenty-four  hours  at  the  body 
temperature.  Streptococci,  staphylococci,  pneumococci,  and  the 
rarer  organisms,  will  probably  have  developed  by  this  time,  and 
will  have  formed  colonies  such  as  have  been  previously  described. 
Weichselbaum's  diplococcus  forms  (on  blood-serum)  "  round, 
whitish,  shining,  viscid-looking  colonies,  with  smooth,  sharply 
defined  outlines  which  attain  a  diameter  of  i  to  if  millimetres 
in  twenty-four  hours."  The  colonies  on  agar  are  similar,  but 
slightly  larger,  and  the  growth  may  become  confluent. 

If  no  colonies  appear  on  blood-serum  or  agar  at  the  end  of 
forty-eight  hours,  the  case  is  probably  due  to  the  tubercle  bacillus 
or  the  gonococcus.  In  some  cases  of  cerebro-spinal  fever  the 
diplococci  in  the  exudate  are  all  dead,  and  cultures  remain  sterile. 

Interpretation  of  Results. 

The  discovery  of  Weichselbaum's  diplococcus  indicates  that 
the  case  is  one  of  cerebro-spinal  fever.  The  chief  importance  in 
making  the  diagnosis  (apart  from  the  fact  that  it  may  throw  light 
upon  the  occurrence  of  several  cases  of  meningitis  within  a  short 
space  of  time  by  proving  the  existence  of  an  epidemic)  arises  from 
the  fact  that  the  prognosis  is  decidedly  better  than  in  other  forms 
of  meningitis.  Roughly  speaking,  about  40  per  cent,  of  cases 
recover,  though  frequently  with  mental  or  other  defects.  It  is  in 
these  cases  that  repeated  lumbar  puncture  is  of  therapeutic  value  ; 
it  should  be  done  whenever  pressure  symptoms  are  urgent.  In 
these  cases  also  great  benefit  can  be  obtained  from  vaccine  treat- 
ment. The  vaccine  should  be  prepared  from  the  patient's  own 
culture,  and  its  use  carefully  controlled  by  frequent  examinations 
of  the  opsonic  index.  The  method  is,  of  course,  useless  in  very 
rapid  cases,  but  very  good  results  are  obtainable  in  the  more 
chronic  ones.  In  some  the  immediate  benefit  resulting  from  each 
injection  is  most  marked. 

Meningitis  due  to  the  pneumococcus  may  arise  from  dissemina- 
tion from  pneumonia  or  other  pneumonic  lesion,  by  spreading 
from  the  middle  ear,  etc.,  or  may  be  primary.  The  examination 
of  the  exudate  throws  no  light  upon  this  point,  and  the  cause  of 
the  infection  must  be  sought  for  on  ordinary  clinical  lines. 

Tuberculous  meningitis  is  proved  by  the  presence  of  tubercle 
bacilli  in  the  fluid,  and  is  indicated  by  sterile  cultures,  absence  of 


138  CLINICAL    BACTERIOLOGY    AND    H.EMATOLOGY 

bacteria  from  the  stained  films,  and  predominance  of  lymphocytes. 
In  these  cases  the  fluid  often  undergoes  a  very  slight  coagulation, 
delicate  cobweb-like  threads  being  observable  after  some  hours. 
This  is  in  itself  strong  evidence  of  tubercle,  and  if  the  deHcate 
coagulum  can  be  withdrawn,  dried  on  a  slide,  and  stained  by 
Ziehl-Neelsen's  method,  there  is  a  fair  chance  of  finding  bacilli 
entangled  with  it.  This  is  not  very  easy  to  do,  the  best  method 
being  to  fish  it  out  with  a  very  fine  piece  of  capillary  tubing  no 
thicker  than  a  pin.  It  is  easy  enough  to  pick  it  out  with  a 
platinum  needle,  but  almost  impossible  to  get  it  off  the  latter  on 
to  the  slide. 

The  other  varieties  of  meningitis  do  not  call  for  special  mention. 

The  chief  value  of  lumbar  puncture  to  the  surgeon  is  that  it 
enables  him  to  diagnose  a  concomitant  meningitis  (indicating  the 
uselessness  of  an  operation)  in  cases  of  lateral  sinus  thrombosis 
and  cerebral  abscess.  The  fluid  usually  becomes  bloody  within 
twenty-four  hours  of  a  fracture  of  the  base  of  the  skull  or  lacera- 
tion of  the  brain.  This  may  assist  in  the  diagnosis  of  obscure 
injuries,  or  of  the  cause  of  a  case  of  unconsciousness  in  which  no 
history  can  be  obtained. 

Haemorrhage  into  the  meninges  is  indicated  by  the  withdrawal 
of  blood-stained  fluid,  but  it  must  be  remembered  that  the  first  few 
drops  may  contain  a  small  quantity  of  blood  which  has  entered 
the  needle  during  its  passage  through  the  tissues,  while  the  rest 
is  clear.  Blood-stained  fluid  may  occur  in  meningitis,  and  should 
be  submitted  to  a  full  examination  for  leucocytes  and  bacteria. 


THE  BACTERIOLOGICAL  EXAMINATION  OF  THE 

BLOOD 

The  bacteriological  examination  of  the  blood  is  not  so  important 
as  might  be  thought,  as  it  is  only  in  a  very  few  diseases  that 
pathogenic  bacteria  are  present  in  the  circulation  in  such  quantities 
as  to  render  the  search  for  them  in  the  minute  amounts  which  are 
withdrawn  for  examination  at  all  promising.  The  method  is 
becoming  of  more  importance  daily,  since  promising  results  have 
been  obtained  in  the  treatment  of  septicaemic  diseases  by  means 
of  specific  vaccines,  which,  to  get  the  full  advantage  of  the 
process,  should  be  obtained  from  cultures  of  the  patient's  own 
bacteria. 

The  chief  organisms  which  have  been  found  in  the  blood  are  : 


THE    BACTERIOLOGICAL    EXAMINATION    OF   THE    BLOOD    I39 

I  and  2.  Streptococci  and  Staphylococci. — These  are  found  in  cases 
of  septicaemia,  pyaemia,  ulcerative  endocarditis,  etc. ;  they  always 
indicate  an  extremely  bad  prognosis.  The  chief  importance  which 
attaches  to  the  discovery  of  these  organisms  is  that  it  absolutely 
settles  the  diagnosis  (always  provided  that  there  are  no  errors  in 
technique),  and  that  it  indicates  whether  the  use  of  antistrepto- 
coccic serum  is  advisable  or  not ;  it  is  useless  in  cases  of  septi- 
caemia, etc.,  which  are  not  due  to  streptococci. 

A  word  of  warning  is  necessary  in  the  interpretation  of  results 
which  indicate  that  staphylococci  are  present  in  the  blood.    These 


Fig.  32. — Spirillum  of  Relapsing  Fever. 

organisms  are  constantly  present  in  the  skin,  and  may  be  found  in 
film  preparations  or  in  cultures,  unless  rigid  antiseptic  precautions 
are  taken.  Streptococci  may  also  occur  as  contaminations  of 
cultures,  but  rarely  occur  in  film  specimens. 

3.  Anthrax  Bacilli.  —  These  may  be  detected  with  ease  and 
certainty,  but  they  are  never  found  in  the  blood  until  it  is  too 
late  to  save  the  patient. 

4.  Tubercle  Bacilli. — These  are  only  present  in  very  scanty 
numbers,  and  are  very  difficult  to  detect.  The  diagnosis  of 
miliary  tuberculosis  is  to  be  made  by  other  methods,  chiefly  by 
that  of  exclusion. 

5.  The  pneumococcHs  is  found  in  severe  cases  of  pneumonia 
(probably  it  might  be  found  in  most  cases  if  a  sufficiently  large 
quantity  of  blood  were  examined)  and  in  septicaemia  and  ulcerative 


140  CLINICAL   BACTERIOLOGY   AND    H.EMATOLOGY 

endocarditis  when  due  to  this  organism.  When  found  in  the  blood 
by  ordinary  methods  it  always  indicates  a  bad  prognosis,  and  sug- 
gests the  use  of  antipneumococcic  serum.  The  identification  of 
the  organism  is  easy  and  certain  if  present  in  quantities  sufficient 
for  it  to  be  found  in  films. 

6.  Typhoid  bacilli.     (See  p.  69.) 

7.  The  bacillus  of  glanders  may  be  found  in  acute  cases  of  that 
disease,  but  its  isolation  and  identification  are  matters  for  an 
expert. 

8.  The  infltienza  bacillus  is  present  in  some  or,  according  to  some 
authorities,  all  cases  of  influenza.  It  may  be  searched  for  in  films, 
but  no  importance  should  be  attached  to  a  negative  result. 

9.  The  bacillus  of  plague. — This  organism  is  often  present  in  the 
blood  in  relatively  large  numbers,  and  the  disease  can  usually 
be  diagnosed  after  a  careful  search  through  a  number  of  suitably 
stained  films.  But  the  investigation  of  a  drop  of  fluid  drawn 
from  the  bubo  (if  one  is  present)  permits  of  an  easier  and  earlier 
diagnosis.  The  blood  examination  is  of  most  value  in  the  pul- 
monary and  septicaemic  forms  of  plague. 

10.  The  spirillum  of  relapsing  fever  is  easily  found,  for  it  possesses 
well-marked  characters  and  is  present  in  great  numbers.  The 
diagnosis  of  relapsing  fever  cannot  be  made  until  it  has  been 
demonstrated  (see  Fig.  32).  It  is  best  seen  by  mounting  a  small 
drop  of  blood  between  slide  and  cover-glass,  and  examining  it  in  a 
perfectly  fresh  state,  when  the  spirilla  are  easily  found  from  the 
commotion  they  cause  by  pushing  aside  the  red  corpuscles. 

11.  The  gonococcus  has  been  found  in  the  blood  in  a  few  cases 
of  ulcerative  endocarditis.  Its  detection  by  cultural  methods  is 
very  difficult,  and  the  services  of  a  bacteriological  expert  should 
be  called  in  if  the  characteristic  cocci  are  not  found  in  blood-films 
in  a  case  in  which  the  diagnosis  of  gonorrhoeal  ulcerative  endo- 
carditis is  probable,  as  further  information  upon  this  point  is 
greatly  needed.  We  may  point  out  that  ulcerative  endocarditis, 
septicaemia,  etc.,  supervening  in  the  course  of  an  attack  of  gonor- 
rhoea, are  not  necessarily  due  to  the  gonococcus.  Any  pathogenic 
bacteria  may  enter  through  the  lesion  of  the  mucous  membrane 
which  the  gonococcus  has  caused. 

12.  The  B.  coli  is  present  in  some  cases  of  septicaemia. 


1 


EXAMINATION    FOR    BACTERIA    IN    FILMS  I4I 

EXAMINATION  FOR  BACTERIA  IN  FILMS 

This  is  the  easiest  method  in  which  bacteria  may  be  found  in 
the  blood,  and  it  does  not  require  such  a  rigid  antiseptic  technique 
as  is  necessary  if  cultures  are  to  be  taken.  The  films  are  prepared 
and  fixed  in  one  or  other  of  the  methods  which  will  be  described 
subsequently  (see  p.  198),  the  only  point  worthy  of  notice  being 
that  the  skin  must  be  very  thoroughly  cleaned;  it  may  be  scrubbed 
with  soap  and  a  nail-brush,  using  plenty  of  hot  water.  The  films 
need  not  be  very  thin  and  even. 

The  method  of  staining  will  depend  upon  the  organism  which 
is  likely  to  be  found,  and  more  especially  whether  it  stains  by 
Gram's  method.  This  is  so  important  in  this  connection  that  a 
repetition  of  a  previous  table  will  not  be  out  of  place. 

Gram's   Method. 
Stained.  Unstained. 

Streptococci.  Typhoid  bacilli. 

Staphylococci.  Bacillus  of  glanders. 

Bacillus  of  anthrax.  Bacillus  of  influenza. 

Bacillus  of  tubercle.  Bacillus  of  plague. 

Pneumococcus.  Bacillus  coli. 

Spirillum  of  relapsing  fever. 

Gonococcus. 

If  the  organism  which  is  present  appears  in  the  first  list,  the 
staining  process  is  simply  that  which  we  have  described  previously, 
and  the  organism  will  be  stained  dark  blue  or  violet,  and  the  other 
structures  will  be  unstained. 

If  the  bacteria  which  are  present  in  the  films  do  not  stain  by 
Gram's  method  the  matter  is  more  difficult,  for  any  stain  which 
colours  them  will  colour  the  nuclei  of  the  leucocytes  also.  Jenner's 
stain  may  be  used,  or  the  film  may  be  stained  by  eosin  and  methy- 
lene blue  separately.  The  organisms  will  then  be  stained  blue. 
Carbol  thionin  is  even  more  suitable,  as  the  colour  which  it 
imparts  to  the  nuclei  of  the  leucocytes  is  not  deep  and  the  red 
corpuscles  are  merely  tinged.  This  is  the  strain  which  we  recom- 
mend for  general  use,  and  in  cases  in  which  the  nature  of  the 
organism  (if  one  be  presented)  is  entirely  unknown. 

If  bacteria  are  detected  by  any  of  these  methods  their  nature 
must  be  recognised  by  a  consideration  of  their  morphological 
features  and  staining  reactions. 


142  CLINICAL    BACTERIOLOGY   AND    HEMATOLOGY 

MALARIA 

The  blood  in  a  suspected  case  of  malaria  may  be  examined 
fresh  or  in  stained  films.  Of  these  methods  the  former  is  the 
better,  and  should  be  used  if  possible.  An  examination  of  stained 
specimens  should  also  be  made,  and  is  convenient,  as  it  can  be 
performed  away  from  the  patient  and  at  leisure. 

Fresh  films  are  made  by  touching  a  drop  of  blood  on  the 
patient's  finger  with  the  centre  of  a  perfectly  clean  cover-glass,  so 
as  to  remove  an  extremely  small  quantity  of  blood.  This  cover- 
glass  is  then  allowed  to  fall  on  to  a  clean  slide,  so  that  the  droplet 
of  blood  may  be  spread  out  by  capillary  attraction  and  by  the 
weight  of  the  cover-glass,  just  as  is  the  case  in  the  method  of 
making  blood-films  to  be  described  subsequently.  But  the  slide  is 
not  separated  from  the  cover-glass;  they  are  examined  just  as 


Fig.  33  — Malarial  Parasites  in  the  Blood. 
The  dark  area  shows  the  parasite  as  it  appears  when  stained  with  thionin. 

they  are,  a  ring  of  vaseline  being  painted  round  the  edge  of  the 
cover-glass  to  prevent  evaporation. 

The  specimen  is  examined  with  a  i-inch  objective,  and  a  place 
found  in  which  the  corpuscles  are  spread  in  a  single  layer  ;  this 
part  is  then  searched  thoroughly  with  a  jV-inch  oil-immersion 
lens.  The  parasites  are  seen  as  pale,  irregularly  shaped  bodies 
with  indistinct  margins,  which  occupy  the  interior  of  the  red 
corpuscles,  and  show  amoeboid  movements  of  greater  or  less  rapidity. 
When  the  parasites  are  older  they  occupy  a  larger  space  in  the 
corpuscles,  and  there  are  granules  of  dark  pigment  around  their 
periphery.  These  granules  are  often  the  first  indications  of  the 
presence  of  parasites  in  the  examination  of  an  unstained  specimen. 
At  a  still  later  stage  the  granules  will  be  found  in  the  centre  of 
the  corpuscle  (the  haemoglobin  of  which  is  now  almost  entirely 
removed),  and  the  parasite  will  show  segmentation  into  a  larger 
or  smaller  number  of  spores  by  lines  which  have  a  radial  arrange- 
ment and   give   the  whole  an  appearance  resembling  that   of  a 


MALARIA  143 

marguerite    daisy.     These    are    only    found    when    a    rigor    is 
"mminent. 

Crescents  are  found  in  the  aestivo-autumnal  formfof  malaria ; 
they  are  crescentic  bodies  with  rounded  "  horns,"  and  contain  a" 
ring  of  pigment  granules  in  the  centre.     They  cannot  be  mistaken 
for  anything  else,  and  if  a  single  one  is  found  it  affords  conclusive 
proof  that  the  patient  has  been  infected  with  malaria. 

Films  for  staining  are  made  in  the  ways  already  described,  and 
must  be  thin  and  perfect.  They  may  be  fixed  by  any  of  the 
methods  we  have  recommended,  the  alcohol-ether  and  the  alcohol- 
formalin  methods  being  perhaps  the  best.  They  may  be  stained 
by  Jenner's  stain,  or  by  eosin  and  methylene  blue  used  separately; 
the  parasites  are  stained  pale  blue  and  the  corpuscles  bright  red. 

A  simpler  stain  is  that  recommended  by  Rees  {Practitioner^ 
March,  1901),  involving  the  use  of  carbol  thionin,  prepared  by 
dissolving  1-5  grammes  of  thoinin  in  10  c.c.  of  absolute  alcohol 
and  100  c.c.  of  a  5  per  cent,  solution  of  carbolic  acid.  This  is  to 
be  kept  for  at  least  a  fortnight,  and  diluted  with  four  times  its 
bulk  of  distilled  water  immediately  before  use.  Staining  is  com- 
plete in  about  ten  minutes.  Ordinary  carbol  thionin  answers 
very  well  indeed.  Thionin  stains  the  red  corpuscles  a  faint  green, 
nuclei  blue,  and  the  parasites  an  intense  purple. 

In  a  suspected  case  of  malaria  the  search  should  not  be 
abandoned  in  less  than  half  an  hour,  or,  in  the  case  of  an 
inexperienced  observer,  much  longer. 

A  fuller  description  of  the  parasite  and  the  differences  between 
the  forms  which  are  present  in  the  various  forms  of  the  disease 
is  beyond  the  scope  of  this  work,  and  the  reader  is  referred  to  the 
admirable  special  number  of  the  Practitioner  mentioned  above. 


EXAMINATION  BY  CULTURAL  METHODS 

This  is  a  much  more  difficult  matter.  The  difficulty  arises 
from  the  abundant  bacterial  flora  of  the  skin ;  unless  the  most 
thorough  antiseptic  precautions  have  been  taken  the  results  are 
ahsoMely  useless.  They  are  worse  than  useless — they  are  mis- 
leading. A  case  of  ulcerative  endocarditis,  for  example,  might 
be  due  to  streptococci,  but  might  be  attributed  to  staphylococci  on 
the  strength  of  an  inadequate  bacteriological  examination.  For 
this  reason  we  are  chary  of  recommending  this  method  of 
diagnosis  in  any  hands  other  than  those  of  an  expert,  and  must 


144  CLINICAL    BACTERIOLOGY   AND    HEMATOLOGY 

urge  the  practitioner  not  to  attempt  it  unless  he  is  prepared  to 
carry  it  out  properly  in  the  most  minute  detail.  It  is  in  particular 
absolutely  useless  to  attempt  to  obtain  cultures  with  the  blood 
taken  from  a  skin  puncture ;  it  must  be  drawn  direct  from  a 
vein,  and  a  large  quantity  (not  less  than  i  c.c,  and  preferably 
4  or  5,  or  even  lo)  must  be  employed. 

One  plan  is  to  use  a  hypodermic  needle,  and  to  plunge  it 
directly  into  a  vein.*  The  process  may  be  carried  out  as 
follows : 

Requisites. — i.  An  all-glass  hypodermic  syringe  of  at  least  5  c.c, 
and  better  10  c.c,  capacity,  preferably  furnished  with  platino- 
iridium  needle. 

2.  Means  of  sterilizing  the  above.  I  personally  keep  the 
syringe  always  sterilized  with  carbolic  lotion  (i  in  20).  After  use 
at  any  time  it  is  washed  out  with  the  lotion,  a  little  of  which  is 
allowed  to  remain  in  the  barrel,  which  is  thus  always  sterile. 
Before  use  the  antiseptic  has  to  be  removed,  and  this  is  effected 
by  filling  the  syringe  two  or  three  times  with  sterile  nutrient 
broth,  a  spare  culture-tube  being  taken  for  the  purpose.  This 
gets  rid  of  the  waste  of  time  involved  in  boiling,  and  the  necessity 
for  carrying  cumbrous  sterilizers. 

Another  method  is  to  boil  the  instrument  for  ten  minutes. 

Or  Wright's  method  may  be  adopted.  Oil  is  heated  to  150°  in 
a  metal  capsule,  and  the  syringe  washed  out  two  or  three  times 
with  the  hot  oil.  If  no  thermometer  is  at  hand  the  temperature 
of  the  oil  can  be  estimated  roughly  by  dropping  a  crumb  of  bread 
into  it.  If  the  crumb  gives  off  bubbles,  the  temperature  is  100°  or 
above ;   it  is  turned  brown  at  1 50°. 

3.  Materials  for  sterilizing  the  skin  are  described  under  the 
heading  of  Lumbar  Puncture. 

4.  A  narrow  bandage  or  piece  of  tape. 

5.  A  spirit-lamp  (not  indispensable). 

6.  Culture  Tubes. — On  the  whole  broth  is  the  most  suitable 
media,  and  rather  large  tubes,  containing  about  25  c.c.  of  broth, 
the  best  to  use.  One  or  two  cultures  on  agar  should  also  be 
taken. 

7.  Collodion,  to  be  applied  to  the  puncture  after  the  operation. 
Method. — Apply  the  narrow  bandage  to  the  upper  arm  suffi- 

*  Dr.  Horder  has  applied  this  method  with  great  success,  and  has  fully 
described  his  technique  and  results  in  the  Practitioner,  November,  1905,  to 
which  the  reader  is  referred  for  further  information. 


EXAMINATION    BY    CULTURAL    METHODS  t45 

ciently  tightly  to  obstruct  the  venous  circulation,  but  not  tightly 
enough  to  check  that  in  the  artery ;  if  the  former  do  not  become 
prominent,  the  patient  should  be  made  to  hang  his  arm  down, 
and  to  clench  and  relax  his  fist. 

Select  a  large  vein  in  the  antecubital  fossa,  and  choose,  if 
possible,  one  that  is  superficial  (as  shown  by  its  blue  colour),  not 
merely  prominent,  since  a  deep  vein  may  slip  in  front  of  the 
needle.  Avoid,  if  you  can,  a  vein  lying  near  an  artery.  Proceed 
to  sterilize  the  skin  in  the  ordinary  way. 

Next  take  the  sterilized  syringe  and  steriUze  the  point  of  the 
needle  in  the  flame  of  the  spirit-lamp.     Proceed  to  make  the 


Fig.  34. — Collection  of  Blood  Direct  from  Vein. 

puncture  as  shown  in  the  illustration  (Fig.  34).  Direct  the  point 
of  the  needle  away  from  the  patient's  body,  so  that  it  faces  the 
blood-flow,  and  enter  it  at  a  point  about  \  inch  from  the  vein,  at 
one  or  other  side.  (This  diminishes  the  chance  of  subsequent 
leakage,  and  possibly  of  sepsis,  organisms  picked  up  by  the 
needle  being  wiped  off"  during  the  passage  of  the  latter  through 
the  tissues.)  Press  it  gradually  onwards  until  the  needle  pierces 
the  wall  of  the  vein,  when,  in  most  cases,  the  blood  will  rise  in 
the  syringe,  slowly  pressing  out  the  piston  before  it.  If  it  does 
not  you  may  make  very  gentle  suction  with  the  piston  :  it  must 
not  be  forcible,  or  the  wall  of  the  vein  will  be  sucked  in  and  act 
as  a  valve. 

In  most  cases  the  only  difficulty  to  arise  will  be  in  entering  the 
vein,  which  may  slip  in  front  of  the  needle  if  the  intravenous 

10 


146 


CLINICAL    BACTERIOLOGY    AND    HEMATOLOGY 


pressure  is  low  or  the  instrument  blunt.     In  this  case  the  vein 

may  be  made  tenser  by  gently  massaging  blood  in  it  towards  the 

bandage  and  retaining  it  there  by  a  finger  pressed  on  to  the  vessel. 

As  soon  as  the  requisite  amount  of  blood  has  been  obtained, 

remove  the  bandage  from  the  upper  arm,  and 

then  withdraw  the  needle  ;  if  you  leave  it  on 

longer  there  may  be  a  considerable  amount 

of  haemorrhage  into  the  tissues,  which  does 

no  harm,  but  leaves  an  unsightly  bruise. 

Next  make  the  cultures  as  follows  :  Expel 
nearly  all  the  blood  into  one  of  the  broth 
tubes  and  shake  gently,  and  then  put  about 
I  c.c.  into  the  agar  tube,  and  place  the  latter 
in  an  incHned  position,  so  that  the  blood  will 
clot  in  an  even  film  over  the  surface. 

Seal  the  puncture  in  the  skin  with  col- 
lodion. If  there  is  haemorrhage  into  the 
tissues,  bandage  the  forearm  evenly  from 
below  upwards. 

Undoubtedly  the  simplest  and   best  of  all 
methods    is   that   described    by   James    and 
j|,  :J  Tuttle   {Report    of   the   Presbyterian   Hospital, 

New  York,  1898).  "  A  piece  of  glass  tubing 
4I  inches  in  length,  and  ^  inch  in  diameter,  is 
drawn  out  to  a  tapered  end,  and  ground  to  fit 
the  cap  of  a  rather  fine  hypodermic  needle. 
The  larger  end  of  the  tube  having  been 
stopped  with  a  cotton  plug,  the  whole  is  then 
placed  in  a  larger  tube,  and  both  ends  of  this 
are  similarly  plugged  with  cotton "**"  (Fig.  35). 
**  The  apparatus  is  then  sterilized  by  dry 
heat.  In  using  it  the  inner  tube  with  needle 
attached  is  removed  ;  the  skin  over  one  of  the 


Bacteriological 
Examination. 


Fig.  35.  — Pipette 
for  Collection 
of    Blood     for   most  prominent  veins  of  the  anterior  surface 

of  the  forearm,  near  the  bend  of  the  elbow,  is 
selected,  a  piece  of  rubber  tubing  or  a  few 
turns  of  a  bandage  being  passed  round  the  arm  above  with 
moderate  pressure,  in  order  to  produce  distension  of  vessels.  The 
needle  is  then  plunged  into  the  vessel,  and  generally  blood  begins 
to  flow  by  the  blood-pressure  itself,  but  any  quantity  desired  may 
*  These  may  be  obtained  from  F.  Ash,  Edmund  Street,  Birmingham. 


EXAMINATION    BY   CULTURAL   METHODS  I47 

be  obtained  by  making  gentle  suction,  either  by  applying  the 
mouth  directly  to  the  end  of  the  tube  where  it  is  stopped  with 
cotton,  or  through  the  medium  of  a  small  piece  of  rubber  tubing 
slipped  over  it. 

"  By  the  above  instrument  vein  punctures  have  been  made  in 
about  150  cases  of  a  variety  of  diseases.  At  no  time  was  any 
difficulty  experienced  in  obtaining  the  amount  of  blood  desired, 
which  was  generally  about  i  c.c.  In  a  few  instances  it  was 
necessary  to  try  two  punctures  before  securing  a  free  flow 
through  the  needle  ;  in  no  case  was  there  any  local  reaction 
whatever  at  the  seat  of  puncture,  nor  did  the  patient  complain  of 
pain  and  annoyance." 

I  used  this  method  for  four  years,  and  found  it  by  far  the  best 
for  clinical  work.  The  needle  is  carried  ready  sterilized,  and  no 
time  is  wasted  in  boiling  it,  and  the  whole  process  may  be  per- 
formed in  five  or  ten  minutes.  The  main  disadvantage  is  that 
it  requires  a  special  instrument,  whereas  an  all-glass  exploring 
syringe  should  be  always  available. 

The  advisability  of  employing  some  such  method  in  which 
the  blood  is  drawn  directly  from  a  vein  in  place  of  the  simple 
skin  puncture  is  very  apparent  from  the  researches  of  Kiihnau 
[Zeitschvift  f.  Hyg.  und  Infct.,  1890),  who  made  parallel  series  of 
experiments  by  the  two  methods.  He  found  that  in  cases  in 
which  the  blood  drawn  directly  from  the  vein  remained  sterile 
growth  (mostly  streptococci  or  staphylococci)  occurred  in  as  many 
as  90  per  cent,  of  cultures  inoculated  from  skin  punctures,  though 
the  most  careful  antiseptic  precautions  were  used. 

The  cultures  thus  obtained  are  incubated  at  the  body  tempera- 
ture, and  examined  from  day  to  day.  The  blood  in  the  broth 
tube  will  coagulate,  and  the  appearance  of  growth  may  be  delayed 
by  the  entanglement  of  the  colonies  in  the  clot ;  sooner  or  later, 
however,  the  clear  fluid  will  become  turbid  if  bacteria  are  present, 
and  subcultures  can  be  made  on  agar  or  blood-serum,  and  films 
examined. 

If  colonies  appear  on  the  agar  tube,  they  are  to  be  carefully 
examined  with  a  lens,  and  their  characters  noticed.  The 
organisms  which  will  be  most  likely  to  develop  are  streptococci, 
staphylococci,  anthrax  bacilli,  pneumococci,  typhoid  bacilli,  the 
bacillus  of  plague,  or  the  B.  coli  ;  the  gonococcus  may  also 
develop,  for  it  will  obtain  the  haemoglobin  necessary  for  its 
development  from  the  blood  itself. 

10 — 2 


148  CLINICAL    BACTERIOLOGY   AND    HEMATOLOGY 

Streptococci  form  small  white  colonies  which  show  no  tendency 
to  run  together  to  form  a  film.  The  centre  of  each  colony  is 
more  opaque  than  its  periphery. 

Staphylococci  form  a  more  or  less  uniform  film,  the  colonies 
extending  laterally  and  fusing  together.  The  growth  is  opaque, 
and  is  of  a  dead  white,  lemon,  or  orange  colour,  according  to  the 
nature  of  the  staphylococcus  present  (albus,  citreus,  or  aureus). 

Anthrax  bacilli  form  small  white  colonies,  having  the  "  barrister's 
wig  "  appearance  already  described. 

The  colonies  of  the  pneiimococcus  are  small  flat  white  points, 
which  do  not  tend  to  fuse  together.  They  are  difficult  to  see 
when  they  are  young,  and,  in  case  of  doubt,  the  tube  should  be 
returned  to  the  incubator. 

The  colonies  of  the  typhoid  bacilli  and  the  B.  coli  are  whitish 
and  opalescent.  They  usually  have  an  angular  or  poly- 
gonal appearance  when  small,  and  tend  to  run  together  when 
older  if  they  are  thickly  set.  Their  discrimination  must  be  left 
to  an  expert. 

The  bacillus  of  plague  forms  white  colonies  which  are  circular  or 
have  a  crenated  outline ;  they  tend  to  run  together,  and  form  a 
uniform  film  over  the  surface  of  the  medium. 

The  gonococcuSf  if  it  develops,  forms  very  minute  transparent 
colonies  which  have  been  compared  to  droplets  of  dew.  They  do 
not  become  confluent.  This  organism  will  not  grow  if  trans- 
planted on  to  the  surface  of  ordinary  media,  unless  a  film  of  blood 
be  previously  spread  over  it. 

After  cultures  have  been  obtained  they  are  to  be  examined 
microscopically  by  the  method  described  on  p.  22,  and  the  morpho- 
logical appearances  compared  with  those  of  the  pathogenic 
organisms  which  we  have  enumerated.  It  is  especially  important 
to  test  whether  the  organism  which  has  been  isolated  stains  by 
Gram's  method  or  not. 


ESTIMATION  OF  THE  OPSONIC  POWER  OF  THE 

BLOOD 

A  very  important  branch  of  blood-work,  and  one  that  seems 
destined  to  be  of  great  value  in  the  future,  has  been  introduced  by 
Wright,  who  has  demonstrated  the  presence  in  the  blood  of  sub- 
stances which  he  calls  opsonins,  and  which  have  the  power  of 
acting  on  pathogenic  bacteria  and  altering  them  so  that  they  can 


ESTIMATION    OF    THE    OPSONIC    POWER    OF   THE    BLOOD    149 

be  taken  up  and  digested  by  leucocytes.  These  substances  are  of 
great  importance  in  that  they  appear  to  be  the  chief  agents  in  the 
production  of  some  forms  of  immunity.  Take,  for  instance,  the 
defence  of  the  body  against  staphylococci.  Leucocytes  have  no 
power  of  taking  up  these  organisms,  and  if  the  protection  of  the 
body  were  entrusted  to  them  alone  a  slight  staphylococcic  lesion 
would  be  a  very  serious  matter.  But  the  blood  contains  a  certain 
amount  of  antistaphylococcic  opsonin — a  greater  amount  in  some 
persons  and  less  in  others — and  this,  by  combining  with  the 
staphylococci,  renders  them  easily  attacked  by  the  leucocytes.  It 
follows  that  where  we  can  measure  the  amount  of  opsonin  present 
we  can  form  some  estimate  of  the  patient's  resisting  power  against 
the  organism  in  question.  It  is  found,  for  instance,  that  the  serum 
of  patients  in  the  early  stages  of  staphylococcic  diseases,  such  as 
pustular  acne  or  boils,  is  usually  very  deficient  in  antistaphylococcic 
opsonins,  whilst  when  cure  takes  place  the  amount  rises  above 
normal.  These  opsonins  are  probably  specific — i.e.,  each  organism 
has  its  own  appropriate  opsonin  :  that  for  tubercle,  for  example, 
is  devoid  of  action  on  staphylococci,  and  vice  versa. 

The  method  given  is  practically  that  used  by  Wright.  It  is  a 
general  method,  and  is  available  for  almost  any  organism,  the  only 
points  of  difference  arising  in  the  preparation  of  the  emulsion  of 
bacteria,  which  differs  somewhat  with  the  various  organisms. 
The  method  is  a  relative  one.  Two  tests  are  made,  one  with  the 
serum  of  the  patient,  and  one  with  that  of  a  healthy  person,  and 
the  results  of  the  two  are  compared  in  the  manner  to  be  described 
subsequently. 

The  process  is  not  altogether  an  easy  one,  and  requires  a  con- 
siderable amount  of  patience  and  some  practice.  Yet  I  know  that 
several  practitioners  have  been  able  to  accomplish  it,  and  as  the 
test  is  of  great  importance  and  interest,  and  as  it  requires  little 
in  the  way  of  apparatus,  it  seems  right  to  give  a  description  of 
it  here. 

The  requisites  are — 

1.  The  serum  of  the  patient  to  be  tested. 

2.  That  of  the  healthy  person  taken  as  a  control. 

These  are  best  collected  in  Wright's  curved  pipettes  (see  p.  34). 
They  must  be  taken  at  approximately  the  same  time  (within  a 
few  hours),  since  the  opsonin  gradually  becomes  inert.  The  test 
should  be  made  not  more  than  three  or  four  days  after  the  blood 
has  been  taken. 


150  CLINICAL    BACTERIOLOGY   AND    HEMATOLOGY 

3.  The  emulsion  of  bacteria.  In  the  case  of  tubercle  an  emul- 
sion of  dead  bacilli  in  normal  saline  solution  is  employed.  This 
can  be  obtained  ready  prepared  from  Messrs.  Allen  and  Hanbury. 
If  you  wish  to  prepare  it  for  yourself,  it  is  necessary  to  take  a 
culture  of  tubercle  bacilli  in  glycerinated  broth  ;  incubate  for  two 
months ;  boil  to  kill  the  bacilli ;  filter  through  filter-paper ;  wash 
with  normal  saline  solution  ;  let  the  bacillary  mass  drain  as  dry 
as  possible,  and  then  place  it  in  a  sterile  tube  and  immerse  in 
boiling  water  for  half  an  hour  to  make  certain  of  its  sterility. 
The  yellowish  mass  thus  obtained  will  keep  indefinitely,  and  will 
serve  for  many  tests.  To  prepare  the  emulsion  from  this,  take  a 
small  portion  (about  as  big  as  a  grain  of  rice)  and  place  it  in  a 
small  agate  mortar,  and  grind  it  up  with  the  pestle  ;  then  add 
normal  saline  drop  by  drop  until  about  2  c.c.  have  been  added, 
continuing  to  grind  meanwhile.  This  gives  an  emulsion  which 
contains  isolated  bacilli  as  well  as  clumps.  These  latter  must  be 
got  rid  of,  and  to  do  this  it  is  necessary  to  centrifugalize  for  three 
or  four  minutes. 

The  staphylococcic  emulsion  is  prepared  by  taking  an  agar 
culture  not  more  than  twenty-four  hours  old,  adding  some  normal 
saline  solution,  and  shaking  gently  so  as  to  wash  off  the  growth. 
When  the  emulsion  is  made  it  must  be  pipetted  off  into  a  small 
tube  and  centrifugalized  for  a  few  minutes.  The  emulsion  must 
not  be  too  thick,  otherwise  the  leucocytes  will  take  up  an  uncount- 
able number  of  cocci ;  the  proper  density  can  only  be  judged  by 
experience,  but  the  emulsion  should  only  be  faintly  opalescent. 
Emulsions  of  pneumococci  and  other  organisms  are  made  in  the 
same  way. 

4.  An  emulsion  of  living  leucocytes.  To  prepare  this  take 
about  10  c.c.  of  normal  saline  solution  containing  J  per  cent,  of 
sodium  citrate,  to  prevent  the  coagulation  of  the  blood.  This  must 
be  freshly  prepared  (or  kept  sterile,  which  is  inconvenient),  and 
the  simplest  method  is  to  use  "  soloids  "  prepared  for  the  purpose 
by  Messrs.  Burroughs  and  Wellcome  (No.  2,456) ;  one  of  these 
dissolved  in  10  c.c.  of  water  will  yield  the  solution  required.* 
This  is  put  into  a  centrifugalizing-tube  and  warmed  to  blood-heat. 
A  healthy  person  is  then  pricked  in  the  ear  or  finger,  and  his 
blood  is  allowed  to  drop  into  the  fluid  until  i  c.c.  or  more  has 
been  collected.  This  is  then  put  into  the  centrifuge,  very  exactly 
counterbalanced,  and  centrifugalized  until  all  the  corpuscles  have 

*  This  very  convenient  method  was  suggested  by  Dr.  Whitfield. 


ESTIMATION    OF   THE    OPSONIC    POWER    OF   THE    BLOOD    15I 

come  to  the  bottom  and  the  supernatant  fluid  is  left  clear.  If  the 
deposit  is  closely  examined  the  red  corpuscles  will  be  seen  to  be 
at  the  bottom,  whilst  above  them  there  is  a  thin  whitish  layer  of 
leucocytes.  Then,  with  a  capillary  pipette  armed  with  an  india- 
rubber  nipple,  the  whole  of  the  clear  fluid  is  to  be  pipetted  off"  as~ 
close  as  possible  to  the  leucocyte  layer,  but  without  disturbing  the 
latter.  Next,  this  layer  and  the  upper  quarter  or  so  of  the  column 
of  red  corpuscles  (which  also  contain  leucocytes)  are  to  be 
pipetted  off  and  put  into  a  small  tube,  and  thoroughly  mixed 
together  by  repeatedly  sucking  them  into  the  pipette  and  then 
expelling  them.  The  result  is  an  emulsion  of  living  leucocytes 
mixed  with  red  corpuscles.  The  presence  of  the  latter  is  a  decided 
advantage.  (It  is  advantageous,  though  not  absolutely  necessary, 
to  rewash  the  leucocytes  in  saline  solution  in  order  to  get  rid  of 
the  citrate  of  sodium.) 

5.  Two  Wright's  pipettes.  These  are  drawn  out  from  a  piece 
of  ordinary  glass  tubing  about  4  inches  long  and  about  as  thick 
as  a  lead-pencil.  This  is  held  at  each  end,  and  the  central  portion 
is  thoroughly  softened  in  a  Bunsen  or  blowpipe  flame,  the  tube 
being  turned  round  the  while.  When  quite  soft  the  tube  is 
removed  from  the  flame,  and  the  ends  then  pulled  firmly  and 
steadily  apart  until  the  softened  portion  is  pulled  out  into  a  thin 
tube  (about  the  thickness  of  a  steel  knitting-needle  or  a  little  less, 
and  a  foot  or  more  long).  This  will  give  two  pipettes,  and  to 
separate  them  melt  the  central  portion  in  a  small  flame,  such  as 
that  of  a  wax  vesta,  and  when  the  glass  is  softened  pull  them 
quickly  apart.     The  whole  pipette  should  be  like  this — 


Fig.  36. 

The  lower  figure  represents  the  point,  which  must  have  as 
nearly  as  possible  the  shape  represented  in  the  figure.  The  ease 
and  accuracy  of  the  process  depends  in  great  measure  on  this 
being  the  case. 

The  Process. — i.  Prepare  a  pipette  by  placing  an  indiarubber 
nipple  on  the  thick  end.  Then  with  a  grease  pencil  or  with  pen 
and  ink  make  a  transverse  line  about  i  inch  from  the  pointed  end. 


152  CLINICAL    BACTERIOLOGY    AND    HEMATOLOGY 

The  volume  of  fluid  contained  in  the  tube  between  the  point  and 
this  mark  is  spoken  of  as  the  unit. 

2.  Having  the  patient's  serum,  the  emulsion  of  leucocytes,  and 
the  emulsion  of  bacteria,  ready  in  front  of  you,  take  the  pipette 
between  the  index-finger  and  thumb  of  the  right  hand,  and  com- 
press the  nipple.  Immerse  the  point  beneath  the  surface  of  the 
emulsion  of  bacilli  and  relax  the  pressure  on  the  nipple  until  the 
emulsion  has  risen  exactly  to  the  mark,  so  that  you  have  drawn 
up  one  unit ;  then  remove  the  point  from  the  fluid  and  relax  the 
pressure  again,  so  that  a  small  column  of  air  will  be  sucked  up. 
This  will  be  quite  easy  if  the  point  is  a  good  one ;  otherwise  it  will 
be  difficult  or  impossible,  as  the  column  of  fluid  will  either  refuse 
to  stir  or  will  oscillate  violently. 

Next  immerse  the  point  in  the  emulsion  of  leucocytes  and  draw 


Fig.  37. 


Fig.  38. 

up  one  unit.  This  will  be  separated  from  the  emulsion  of  bacteria 
by  the  short  column  of  air.  Remove  the  point  from  the  emulsion 
and  draw  up  a  second  column  of  air ;  reinsert  it  and  draw  up 
a  second  unit  of  leucocytes,  and  then  a  third  column  of  air. 

Lastly,  draw  up  one  unit  of  the  serum.  You  will  then  have  in 
your  pipette  (counting  from  the  nipple  towards  the  point)  one  unit 
of  bacterial  emulsion,  a  column  of  air,  a  unit  of  leucocytes,  a 
column  of  air,  a  second  unit  of  leucocytes,  a  column  of  air,  and, 
lastly,  a  unit  of  serum  (Fig.  37)." 

3.  Put  the  point  of  the  pipette  on  to  a  clean  slide  and  express 
the  whole  of  its  contents,  and  mix  them  well  together,  sucking 
them  repeatedly  into  the  pipette  and  expelling  them.  When 
thoroughly  mixed  suck  them  into  the  pipette,  suck  up  a  short 
column  of  air,  and  seal  the  tip  in  the  flame  (Fig.  38). 

Then  place  the  pipette  point  downwards  in  the  incubator  at 
35°  to  37°  ^-j  noting  the  time  exactly,  and  proceed  to  prepare  a 
second  pipette  in  exactly  the  same  way,  using  the  same  emulsions 

*  It  is  not  necessary  to  take  more  than  one  unit,  and  where  many  estimations 
are  being  made  one  only  should  be  employed,  as  less  ' '  cream  ' '  is  required. 


ESTIMATION   OF   THE    OPSONIC    POWER    OF   THE    BLOOD    I53 

of  bacteria  and  leucocytes,  but  the  control  serum  instead  of  the 
patient's.  Place  this  in  the  incubator  by  the  side  of  the  other, 
noting  the  time  at  which  you  do  so.  When  no  incubator  is  at 
hand  the  tubes  may  be  placed  in  a  vessel  of  water,  which  can  be 
kept  at  blood-heat  for  the  necessary  time  (fifteen  minutes)  witlT 
very  little  trouble,  or  the  Dewar  flask  mentioned  on  p.  21  may  be 
used. 

When  each  pipette  has  been  incubated  for  a  quarter  of  an 
hour,  remove  it  from  the  incubator,  break  off  the  end,  fit  the 
nipple  to  the  thick  end,  and  expel  the  contents  on  to  a  clean 
slide.  Next  mix  them  thoroughly  together.  Then  prepare  suit- 
able films  in  the  usual  manner.  The  best  method  for  this  is 
the  one  given  on  p.  198,  in  which  the  film  is  spread  on  two  cover- 
glasses.  A  small  drop  of  the  mixture  is  placed  on  one  of  the 
cover-glasses  held  in  the  left  hand  in  the  manner  described,  the 
second  glass  applied,  and  the  two  slid  apart.  The  process  is  very 
easy,  and  if  the  cover-glasses  have  been  properly  cleaned  two 
excellent  films  will  result. 

Most  bacteriologists  spread  their  films  on  slides,  using  a  second 
slide  as  a  spreader.  I  am  convinced  that  this  method  is  not  so 
good  as  that  given  above.  It  is  true  that  it  renders  the  counting 
somewhat  easier,  since  the  leucocytes  are  all  collected  together  at 
the  end  of  the  film,  whereas  with  the  cover-glass  method  they  are 
scattered  all  over  the  surface.  But  the  counts  by  the  latter 
method  are  much  more  uniform,  the  leucocytes  more  defined,  and 
there  is  less  danger  of  bacilli  being  ground  mechanically  into  the 
cells ;  there  is  always  a  considerable  margin  of  error  in  the  pro- 
cess, and  I  believe  that  the  extra  amount  of  accuracy  obtainable 
in  this  way  is  quite  worth  the  additional  trouble  involved. 

The  films  have  next  to  be  stained.  When  the  organism  in 
use  is  the  staphylococcus,  pneumococcus,  etc.,  Jenner's  stain  is  as 
good  as  any  ;  or  the  film  may  be  fixed  with  formalin  (p.  203)  or 
perchloride  of  mercury  (p.  203),  and  stained  with  carbol  thionin. 
In  the  case  of  tubercle  bacilli  it  is  best  to  fix  with  saturated  solu- 
tion of  perchloride  of  mercury  (one  or  two  minutes),  wash,  stain 
in  the  ordinary  way  with  hot  carbol  fiichsin,  decolorize  for  half  to 
one  minute  in  2^  per  cent,  sulphuric  acid  in  methylated  spirit,  and 
to  counterstain  for  about  five  minutes  in  borax  methylene  blue, 
or  in  Delafield's  haematoxylin.  It  is  necessary  to  get  the  proto- 
plasm of  the  leucocytes  clearly  defined,  so  that  a  powerful  stain 
is  necessary.     Washj  dry,  mount. 


154  CLINICAL    BACTERIOLOGY    AND    HEMATOLOGY 

Lastly,  the  films  are  examined  with  the  oil-immersion  lens.  The 
polynuclear  leucocytes  will  be  found  to  contain  the  bacteria,  and 
it  will  be  necessary  to  count  the  number  in  each  of  fifty  leucocytes 
in  both  your  preparations — i.e.,  in  that  made  with  the  patient's 
serum  and  in  the  control  made  with  that  from  a  healthy  person. 
The  ratio  between  the  two  gives  the  opsonic  index.  For  example, 
in  one  case  the  number  of  tubercle  bacilli  contained  in  fifty  poly- 
nuclear leucocytes  taken  at  random  amounted  to  78.  In  the 
control  specimen  from  a  healthy  person  the  same  number  of  poly- 
nuclears  contained  172.  The  ratio  xV%  =  o*45  gives  the  opsonic 
index  ;  it  shows  that  the  patient  has  less  than  half  the  normal 
amount  of  opsonin,  and  probably,  therefore,  less  than  half  the 
normal  resisting  power  to  the  tubercle  bacillus. 

The  diagnostic  value  of  the  examination  is  considerable.  In 
acute  infections  the  index  is  almost  always  low  to  the  organism 
causing  the  disease,  but  normal  to  others.  For  example,  in 
a  case  of  severe  furunculosis  the  index  to  staphylococci  was 
0-65,  and  in  a  case  of  pustular  acne  07.  In  a  case  of  septicaemia 
due  to  streptococci  the  index  (tested  with  a  culture  of  strepto- 
cocci obtained  from  the  patient's  blood)  was  o-6  on  several 
occasions. 

When  spontaneous  cure  takes  place  it  is  accompanied  by,  and 
is  apparently  due  to,  an  increase  of  the  opsonic  power  of  the  blood. 
Thus,  in  pneumonia  the  opsonic  index  tested  with  pneumococci  is 
below  normal  until  the  crisis  is  reached,  when  there  is  a  sudden 
rise  above  normal,  so  that  the  patient  is  for  a  short  period  more 
resistant  than  the  healthy  person.  In  staphylococcic  lesions  the 
rise  is  as  a  rule  more  gradual  and  irregular,  and  the  lesions  may 
persist  when  the  index  is  high,  though  in  most  cases  this  heralds  a 
rapid  improvement. 

The  behaviour  of  the  opsonic  index  in  tuberculosis  is  very  in- 
teresting. The  main  feature  is  that  it  is  very  variable,  especially 
in  patients  in  whom  the  disease  is  progressing  rapidly  and  in  those 
who  are  taking  exercise.  This  variability  is  supposed  to  be  due 
to  auto-inoculation,  bacilli  being  detached  from  the  lesions  and 
lodged  in  the  tissues,  where  they  act  as  small  doses  of  vaccine. 
As  a  general  rule  it  is  thought  that  (in  lupus  especially"^)  a  high 
index  is  of  favourable  import,  and  vice  versa,  but  there  is  not  the 
same  more  or  less  direct  relation  between  a  high  index  and  the 
process  of  cure  that  there  is  in  pneumococcic  and  some  other 
*  Bulloch,  Trans.  Path.  Soc,  1905. 


ESTIMATION    OF    THE    OPSONIC    POWER    OF    THE    BLOOD    155 

infective  processes.  A  patient  may  die  of  tuberculosis  whilst  his 
opsonic  index  is  high.  This  seems  paradoxical,  but  the  conditions 
are  exceedingly  complex,  and  the  symptoms  of  pulmonary  tuber- 
culosis are  due  almost  as  much  to  the  other  organisms  (strepto- 
cocci, etc.)  as  to  the  tubercle  bacilli  itself.  In  miliary  tuberculosis 
the  index  is  often  high,  and  shows  great  variations  in  a  short  time. 
I  believe  you  are  almost  safe  in  diagnosing  tubercle  if  the  index 
is  below  0-8  or  above  1*2,  and  if  it  shows  marked  variations  from 
day  to  day  the  probability  is  still  greater. 

A  low  opsonic  index  towards  a  given  organism,  therefore, 
denotes  either  (i)  an  infection  with  that  organism,  or  (2)  a  low 
power  of  resistance,  so  that  if  the  patient  is  exposed  to  infection 
invasion  will  readily  take  place.  In  such  cases  he  should  be  care- 
fully shielded  from  exposure,  and  the  general  health  improved  by 
fresh  air,  careful  feeding,  tonics,  etc. 

A  high  opsonic  index  {i.e.,  one  decidedly  above  normal)  usually 
indicates  that  the  patient  has  had  an  attack  of  the  disease  caused 
by  the  organism  in  question,  and  has  overcome  it.  Normal 
persons  differ  very  little  amongst  themselves  ;  for  instance,  in  a 
series  of  healthy  persons,  if  the  average  index  be  taken  as  normal, 
it  is  unusual  to  find  one  below  0-95  or  above  i'05. 

A  study  of  the  opsonic  index  is  the  basis  of  Wright's  method 
of  treatment  by  means  of  vaccines.  In  carrying  this  out  the 
patient's  opsonic  index  is  raised  by  injections  of  the  organisms 
which  cause  the  disease  (carefully  sterilized)  in  appropriate  doses. 
These  are  given  in  a  healthy  part  of  the  body,  where  the  tissues 
are  stimulated  to  produce  opsonins  against  the  organisms  intro- 
duced, and  these  opsonins  are  carried  in  the  blood  to  the  lesion. 
The  reason  why  it  is  necessary  to  estimate  the  opsonic  index  of 
the  blood  from  time  to  time  in  carrying  out  this  process  is  that 
the  injection  causes  it  to  fall  for  a  variable  time  (usually  a  few 
days — the  negative  phase),  and  if  a  second  injection  be  given 
before  this  fall  has  gone  off  and  been  succeeded  by  a  rise  above 
the  initial  level,  harm  rather  than  good  will  follow. 

The  method  of  preparing  these  vaccines  is  roughly  as  follows  : 
The  cultures  should  in  all  cases  where  practicable  be  prepared 
from  the  organisms  isolated  from  the  patient  himself.  "Stock" 
vaccines  may  do  good  in  some  cases,  especially,  perhaps,  in 
staphylococcic  diseases  ;  but  in  diseases  due  to  streptococci,  B,  coli, 
etc.,  they  are  not  nearly  so  likely  to  succeed  as  vaccines  specially 
prepared :  these  organisms  differ  slightly  in  different  cultures,  and 


156  CLINICAL    BACTERIOLOGY    AND  HEMATOLOGY 

a  vaccine  prepared  from  one  variety  may  have  little  action  on 
another.  The  culture  should  be  made  on  agar  (or  blood-agar  in 
the  case  of  gonococci,  etc.),  and  should  be  one  or  two  days  old. 
To  each  culture- tube  about  2  to  5  c.c.  of  sterile  normal  saline 
solution  are  added,  and  the  bacteria  scraped  off  by  means  of  a 
platinum  needle  and  the  tube  gently  shaken,  so  that  the  organisms 
are  emulsified.  The  number  of  bacteria  in  this  emulsion  must 
next  be  counted.  Wright's  method  is  usually  employed,  and  is 
carried  out  by  mixing  the  bacterial  suspension  with  blood  in  known 
proportions,  making  films,  and  counting  the  red  corpuscles  and 
bacteria  in  the  same  field  of  the  microscope,  so  as  to  obtain  the 
relative  proportions  of  the  two.  The  number  of  red  corpuscles 
per  cubic  millimetre  is  known  (or  can  be  counted),  and  from  that 
the  number  of  organisms  can  be  calculated.  Proceed  as  follows  : 
Take  an  opsonin  pipette,  and  draw  up  one  volume  of  blood  from 
a  healthy  person,  then  a  bubble  of  air,  then  one  volume  of  citrate 
solution  (p.  150),  then  one  volume  of  the  emulsion.  Mix  them 
together  on  a  slide,  make  films  on  two  cover-glasses,  dry,  and 
stain  by  Jenner's  method.  Proceed  to  count  the  red  corpuscles 
and  bacteria  on  several  fields  of  the  microscope  :  this  will  be  easier 
if  you  rule  four  ink  lines  enclosing  a  square  on  the  lower  lens  of 
your  eyepiece,  and  count  the  objects  lying  therein.  When  you 
have  done  this,  move  the  slide  so  as  to  get  a  fresh  film,  and  count 
again.  Do  this  a  large  number  of  times,  and  add  the  totals  of  the 
corpuscles  and  of  the  bacteria,  and  calculate  the  ratio  between  the 
two.  (Thus  in  one  case  the  total  red  corpuscles  on  the  various 
fields  were  18,  15,  20,  21,  14,  17,  10,  and  15  ;  and  the  bacteria  37, 
26,  31,  40,  25,  30,  32,  36  :  the  totals  were  130  and  257,  or,  roughly, 
I  to  2.)  The  calculation  of  the  number  of  bacteria  per  cubic 
millimetre  is  then  easy,  and  that  multiplied  by  1,000  gives  the 
number  per  c.c. 

Next  the  emulsion  has  to  be  sterilized  by  heat.  It  is  placed  in 
a  test-tube,  which  is  drawn  out  and  sealed  in  a  blow-pipe  flame 
and  completely  immersed  in  a  water-bath  at  60°.  After  one  hour  it 
is  removed,  and  a  small  amount  placed  on  a  suitable  culture 
medium  and  incubated  (in  order  to  ascertain  its  sterility),  and  the 
tube  re-sealed.  If  sterile,  it  is  now  to  be  diluted  with  a  0-25  per 
cent,  solution  of  lysol  or  carbolic  acid  in  sterile  normal  saline 
solution  to  such  an  extent  that  the  dose  required  is  made 
I  c.c.  Thus  an  emulsion  of  staphylococci  was  found  to  contain 
2,500,000,000  cocci  per  c.c.     The  dose  required  was  500,000,000, 


ESTIMATION    OF   THE    OPSONIC    POWER   OF   THE    BLOOD    I57 

SO  that  I  part  of  the  emulsion  was  diluted  with  four  of  0*25  per  cent. 
lysol.  Lastly,  it  is  pipetted  off  with  a  i  c.c.  pipette  or  hypodermic 
needle  (of  course,  sterile)  into  i  c.c.  ampoules,  previously  sterilized 
by  heat. 

The  dosage  varies  with  different  bacteria  and  with  differeril 
cultures,  and  the  following  are  approximate  only.  With  staphy- 
lococci it  varies  between  50,000,000  and  1,000,000,000, 500,000,000 
being  a  good  average.  Meningococci  may  be  given  in  the  same 
doses,  or  rather  smaller  :  with  a  young  child  I  commence  with 
25,000,000.  B,  coli  is  usually  rather  toxic,  and  may  cause  a  good 
deal  of  local  and  general  reaction,  and  the  first  dose  should  not 
exceed  20,000,000 ;  it  may  subsequently  be  increased  somewhat 
if  thought  necessary.  With  pneumococci  the  dose  may  be 
50,000,000  to  100,000,000  or  more,  and  with  gonococci  50,000,000 
to  250,000,000. 

The  dose  of  tuberculin  (T.  E.  or  T.  R.)  is  y^L^  milligramme  to 
jjqL^  milligramme.  The  material  is  bought  in  bottles  containing 
I  to  5  C.C.,  with  the  strength  stated  on  the  label,  and  dilutions 
prepared  with  0*25  per  cent,  normal  saline  solution,  previously 
sterilized. 

As  regards  the  question  as  to  the  necessity  of  controlHng  the 
injections  by  observations  of  the  opsonic  index,  it  is  difficult  to 
speak  with  confidence.  With  staphylococci  it  is  probably  un- 
necessary, and  a  small  dose  every  week,  or  a  larger  one  every 
fortnight,  will  usually  yield  good  results.  In  the  case  of  tubercle 
I  am  doubtful  as  to  its  advantage,  but  usually  carry  it  out  for  one 
or  two  injections  in  order  to  make  sure  that  I  am  not  dealing  with 
a  patient  with  a  very  long  negative  phase,  in  which  case  I  lower 
the  dose.  In  the  other  organisms  I  prefer,  if  possible,  to  take  the 
index  regularly,  and  I  believe  this  is  especially  desirable  in  the 
case  of  meningococcic  infections. 

The  results  of  this  method  of  treatment  are  extremely  good 
in  the  case  of  staphylococcic  lesions  (boils,  acne,  etc.),  though 
in  some  chronic  cases  a  long  course  is  necessary.  In  the 
complications  and  sequelae  of  gonorrhoea  (iritis,  arthritis,  etc.) 
great  benefit  is  also  obtained,  and  some  chronic  pneumococcic 
lesions  (such  as  sinuses  left  after  empyemata)  heal  very  rapidly 
after  one  or  two  injections.  Chronic  cystitis  and  pyelitis  due  to 
B.  coli  are  sometimes  cured,  but  more  often  the  symptoms  are 
greatly  reUeved  without'  actual  cure  being  obtained.  Some  cases 
of  ulcerative  endocarditis  and  septicaemia  have  been  completely 


158  CLINICAL    BACTERIOLOGY    AND    H.^MATOLOGY 

cured,  and  the  other  applications  of  the  process  are  too  numerous 
to  mention  here.  In  general  we  may  say  that  it  should  be  tried 
in  all  chronic  infective  diseases  that  do  not  yield  readily  to  easier 
methods. 

In  tuberculosis  the  results  have  been  on  the  whole  disappointing, 
and  the  method  should  be  reserved  as  an  adjuvant  to  other 
treatment,  or  for  lesions  (tuberculous  iritis,  ulceration  of  the 
bladder,  etc.)  in  which  direct  surgical  treatment  is  unavailable. 
Tuberculous  sinuses  often  heal  well  after  a  few  injections,  and 
tuberculous  ulceration  of  the  skin  does  better  than  ordinary  lupus. 


COLLECTION  OF  MATERIAL  AT  POST-MORTEM 
EXAMINATIONS 

The  saprophytic  bacteria  which  occur  in  such  vast  numbers  in 
the  skin  and  alimentary  canal  during  life  undergo  very  rapid 
multiplication  after  death ;  hence,  in  cases  where  bacteriological 
examinations  have  to  be  made,  the  sectio  should  be  performed  as 
soon  as  possible  after  death. 

The  materials  which  should  be  examined  in  all  cases  are  the 
heart-blood,  the  spleen,  and  the  liver,  and  the  following  methods 
are  to  be  employed  : 

The  heart-blood  should  be  collected  in  the  method  which  has 
been  described  previously  (see  p.  33),  and  cultures  may  be  made 
upon  the  spot,  or  the  pipettes  sealed  at  both  ends  and  taken  to  the 
laboratory. 

The  spleen  may  usually  be  examined  in  the  same  way.  If  it  is 
so  firm  and  hard  that  no  fluid  rises  into  the  pipette,  it  should  be 
treated  in  the  same  way  as  the  liver. 

Cultivations  should  be  made  from  the  liver  at  the  time  when  the 
autopsy  is  performed.  The  organ  should  be  cut  in  half,  and  a 
small  portion  of  the  cut  surface  deeply  seared  with  a  hot  iron. 
This  area  is  then  to  be  perforated  with  a  stout  platinum  needle, 
and  the  culture  media  inoculated  at  once. 

If  the  material  has  to  be  taken  to  a  distance,  and  no  culture- 
tubes  are  at  hand,  a  different  course  must  be  adopted .  The  simplest 
way  is  to  cut  out  a  cube  of  liver  substance  from  the  centre  of  the 
organ,  and  to  sear  every  part  of  its  surface^  with  the  fiat  of  a  red- 
hot  knife.  The  block  (which  may  be  about  as  large  as  a  lump  of 
sugar)  must  be  dropped  at  once  into  a  sterilized  bottle.     Another 


SECTION-CUTTING  I59 

plan  is  to  sear  the  surface  of  the  block,  and  then  to  tie  a  piece  of 
string  round  it  and  dip  it  quickly  into  melted  paraffin  (a  candle 
will  do),  and  allow  the  coating  to  set ;  the  dipping  is  to  be 
repeated  several  times,  and  the  specimen  (string  and  all)  may^ 
then  be  packed  without  further  precautions.  In  any  case  it  must 
reach  the  laboratory  as  soon  as  possible. 

Where  cultural  examinations  are  not  required,  small  portions 
of  the  organs  should  be  placed  in  a  suitable  hardening  fluid  as 
soon  as  possible.  Equal  parts  of  methylated  spirit  and  water  is 
perhaps  as  good  as  anything,  and,  in  the  absence  of  this,  undiluted 
whisky  or  other  spirit  answers  equally  well  (see  p.  161). 

Other  solid  organs  are  treated  in  the  same  way.  Fluids  (pus, 
the  contents  of  cysts,  pericardial  or  other  fluid,  etc.)  should  be 
collected  in  pipettes  in  the  manner  adopted  for  the  heart-blood. 


SECTION-CUTTING 

The  methods  employed  in  section-cutting  are  somewhat  outside 
the  scope  of  this  work,  inasmuch  as  sections  are  rarely  necessary 
for  the  purposes  of  bacteriological  diagnosis,  and  I  have  attempted 
to  give  the  simplest  possible  methods  in  all  cases.  The  presence 
of  bacteria  in  the  tissues  can  usually  be  demonstrated  by  the  simple 
processes  of  smearing  the  cut  surfaces  of  tissues  on  clean  slides 
or  cover-glasses,  and  treating  the  films  thus  obtained  by  the  fixing 
and  staining  methods  previously  described.  If,  for  instance,  we 
have  to  search  for  tubercle  bacilli  in  tuberculous  glands,  it  is 
usually  sufficient  to  smear  the  cut  surfaces  of  the  glands  on  a  slide, 
dry,  fix  by  heat,  and  stain  in  the  same  way  as  sputum  is  stained 
for  the  tubercle  bacillus.  If  anthrax  bacilli  were  being  looked  for 
in  the  liver  or  other  organ  removed  post-mortem,  the  same 
method  of  procedure  would  be  adopted,  except  that  Gram's 
method  of  staining  would  be  used.  So  also  for  typhoid  bacilli  in 
the  spleen,  where  the  film  would  be  stained  with  a  simple  stain 
such  as  thionin  or  Loffler's  methylene  blue. 

It  seems  advisable,  however,  to  give  a  short  general  account  of 
the  processes  involved  in  section-cutting,  for  they  are  by  no  means 
difficult,  and  do  not  require  very  elaborate  apparatus.  Further, 
the  same  methods  of  section-cutting  are  used  for  investigating 
the  nature  of  tumours,  etc.,  and  this  is  done  already  by  many 
practitioners  and  should  be  done  by  still  more. 


l60  CLINICAL   BACTERIOLOGY   AND    HEMATOLOGY 

Slices  of  organs  or  tissues  which  are  to  be  cut  have  first  to  be 
fixed.  The  process  of  fixation  consists  essentially  in  the  applica- 
tion of  some  agent  which  brings  about  coagulation  of  the  com- 
ponent proteids  with  as  little  distortion  of  the  morphological 
elements  as  possible ;  if  this  step  were  not  carried  out  the 
subsequent  processes  would  be  liable  to  cause  alterations  in  the 
shape,  size,  and  appearance  of  the  cells  and  fibres.  There  are 
two  chief  methods  of  fixation,  that  involving  the  use  of  chemical 
substances,  and  that  involving  the  use  of  heat.  The  processes 
which  are  used  in  fixing  the  tissues  harden  them  at  the  same  time  ; 
this  is  necessary,  for  fresh  tissue  would  yield  before  the  sharpest 
knife,  and  could  not  be  cut  into  thin  sections.  These  processes  are 
always  carried  out,  no  matter  what  method  of  section  cutting  is  to 
be  adopted. 

In  cutting  sections  it  is  necessary  that  the  material  should  be 
sufficiently  firm  and  homogeneous  in  consistency.  The  former  is 
secured  to  some  extent  by  the  process  of  hardening,  but  a 
properly  hardened  block  is  rarely  firm  enough  to  permit  of  its 
being  cut  into  sections  without  further  preparation.  Further,  it 
almost  invariably  happens  that  some  parts  of  the  material  are 
firmer  or  harder  than  others  ;  and  if  such  a  substance  were  cut 
the  harder  parts  might  be  sufficiently  firm,  whilst  the  softer  parts 
would  simply  crumble  before  the  knife.  There  are  two  methods 
of  overcoming  this  difficulty — freezing  and  embedding. 

The  freezing  process  is  very  simple,  and  it  is  one  which  can  easily 
be  carried  out  at  home.  The  sections  which  it  yields  are  usually 
quite  sufficient  for  purposes  of  histological  research  (the  diagnosis 
of  tumours,  etc.),  but  they  are  rarely  sufficiently  thin  for  a  proper 
demonstration  of  the  bacteria  which  they  may  contain.  The 
sections  are  cut  more  easily  by  the  freezing  than  by  the  paraffin 
process,  but  they  are  decidedly  more  difficult  to  manipulate. 

In  the  freezing  process  the  block  or  tissue  after  fixing  and 
hardening  is  dipped,  or,  better,  soaked  for  some  hours,  in  a  thick 
solution  of  gum  arable.  It  is  then  placed  on  the  plate  of  a  micro- 
tome and  frozen  until  the  tissue  assumes  the  consistency  of  fairly 
hard  cheese,  and  can  be  cut  into  thin  sections. 

The  embedding  process  should  be  called  the  infiltration  process  ; 
the  tissue  to  be  cut  is  infiltrated  throughout  with  some  firm 
substance,  and  not  merely  embedded  therein.  Two  embedding 
materials  are  in  general  use — paraffin  and  celloidin.  The  latter 
will  not  be  described,  as  it  is  only  necessary  for  special  work,  and 


FIXING    MATERIAL   FOR   CUTTING  l6l 

for  ordinary  purposes  cannot  compare  with  paraffin  for  beauty  of 
results  and  facility  of  application. 

In  the  paraffin  process  the  tissue  is  infiltrated  throughout  with 
hard  paraffin  (such  as  is  used  for  the  better  varieties  of  paraffin 
candles),  so  that  every  cell  and  every  fibre  is  permeated  and 
supported  on  every  side.  To  do  this  requires  a  number  of 
processes.  It  would  be  of  no  use  to  immerse  the  block  of  tissue 
in  the  paraffin  just  as  it  is,  for  the  paraffin  would  not  wet  it,  much 
less  soak  into  it.  The  water  is  first  removed;  and  this  is  done  by 
soaking  the  material  in  absolute  alcohol.  But  alcohol  does  not 
-dissolve  paraffin  or  mix  with  it ;  it  is  therefore  necessary  to  remove 
it  by  means  of  some  fluid  which  will  mix  with  it  on  the  one  hand 
and  paraffin  on  the  other.  Of  these  there  are  many  :  xylol, 
chloroform,  benzine,  cedar  oil,  and  many  more  are  in  use  for 
special  purposes.  Chloroform  answers  most  purposes,  and  is  to 
be  generally  recommended.  The  block  of  tissue  is  now  ready  to 
be  soaked  in  melted  paraffin  ;  it  is  kept  in  a  bath  of  this  substance 
until  the  chloroform  has  been  entirely  driven  off  and  replaced  by 
the  paraffin.  The  whole  is  then  allowed  to  cool,  is  shaped  into 
suitable  blocks,  and  is  then  ready  for  cutting. 

We  shall  now  describe  the  processes  in  fuller  detail. 


FIXING  MATERIAL  FOR  CUTTING 

These  processes  must  be  understood  by  all  practitioners,  even 
although  they  do  not  intend  to  cut  sections  for  themselves.  It 
happens  to  every  medical  man  to  find  it  necessary  to  send  tumours, 
etc.,  to  a  laboratory  to  obtain  a  pathological  diagnosis ;  and  in 
very  many  cases  the  materials  are  treated  in  a  way  which 
absolutely  prevents  good  sections  being  obtained.  Many  fixing 
fluids  are  in  use,  and  any  of  them  may  be  selected,  but  it  is 
absolutely  necessary  that  the  material  to  be  investigated  should  be 
cut  into  small  pieces  and  put  into  a  large  bulk  of  the  fluid  at  once. 
This  is  especially  necessary  in  the  case  of  material  removed  at  a 
post-mortem  examination,  where  the  tissues  and  organs  have 
already  undergone  alteration. 

As  regards  the  size  of  the  slices  which  are  to  be  placed  in  the 
hardening  fluid,  it  is  sufficent  to  say  that  they  should  never 
exceed  -^  inch  in  thickness,  and  if  perchloride  of  mercury  is  used 
should  be  even  thinner.  The  other  dimensions  of  the  block  are 
of  less  importance. 

II 


ibz  CLINICAL    BACTERIOLOGY   AND    HEMATOLOGY 

The  bulk  of  the  fluid  in  which  the  block  is  placed  should  be  at 
least  twenty  times  that  of  the  block,  and  it  is  not  advisable  to 
place  two  blocks  in  the  same  vessel. 

The  fluids  which  we  shall  recommend  for  this  purpose  are  : 

1.  Perchloride  of  mercury  in  normal  saline  solution.  This  is 
prepared  by  dissolving  common  salt  in  water  in  the  proportion  of 
7  grammes  to  a  litre  (about  3 J  grains  to  the  ounce),  and  saturating 
this  solution  whilst  hot  with  perchloride  of  mercury.  The 
solution  must  be  allowed  to  cool  completely ;  as  it  does  so, 
crystals  of  the  mercury  salt  will  separate  out. 

This  fluid  fixes  completely  in  twenty-four  hours,  or  less,  and 
gives  most  excellent  results.  Its  powers  of  penetration  are  not 
very  great,  so  that  slices  of  tissue  which  are  to  be  fixed  in  it 
should  be  thin. 

The  after-treatment  of  the  blocks  fixed  in  this  fluid  must  be 
described  briefly.  They  are  allowed  to  remain  in  the  solution  for 
twenty-four  hours  and  no  longer,  and  are  then  washed  for  twenty- 
four  hours  in  running  water  to  remove  the  perchloride  of  mercury. 
They  are  then  passed  through  the  various  strengths  of  spirit  (as 
will  be  described  subsequently),  a  little  tincture  of  iodine  being 
added  to  each  to  remove  any  mercury  which  may  still  remain. 
The  other  steps  are  the  same  as  those  which  are  used  if  other 
methods  of  fixation  have  been  adopted. 

2.  Formalin.  This  should  be  used  in  a  5  per  cent,  solution  in 
normal  saline  solution  (o'8  per  cent.).  It  yields  very  good  results, 
and  is  perhaps  the  fluid  which  can  be  most  warmly  recommended 
to  a  practitioner  who  is  going  to  send  his  material  to  a  public 
laboratory. ■'^  The  fluid  has  very  great  powers  of  penetration,  and 
the  slices  may  be  much  thicker  than  we  have  recommended. 
The  one  objection  to  the  fluid  is  that  it  interferes  somewhat  with 
the  way  in  which  the  sections  stain. 

3.  Alcohol  is  a  very  good  fixing  fluid.  When  it  is  used  the 
blocks  should  be  cut  small  and  placed  at  once  in  methylated 
spirit  diluted  with  an  equal  quantity  of  water. 

*  Formalin  should  not  be  used  for  tissues  which  are  to  be  searched  for  the 
tubercle  bacillus,  as  it  prevents  the  decolorizing  action  of  the  acid. 


SECTION-CUTTING    BY   THE    FREEZING    METHOD 


163 


SECTION-CUTTING   BY   THE   FREEZING   METHOD 

Sections  which  are  prepared  by  the  freezing  method  are  rarely 
as  thin  as  those  prepared   by   one  or   other   of  the  infiltration- 
processes,  but  are  prepared  very  rapidly,  and  are  often  sufficient 
for  diagnostic  purposes,  where  rapidity  is  the  first  consideration. 

The  blocks  of  tissue  must  be  hardened  before  being  cut,  any 
of  the  above  fluids  being  applicable  ;  where  alcohol  is  used  it 
must  be  washed  out  in  water,  as  it  will  not  freeze.  Where 
more  rapid  work  is  required  the  best  method  is  a  modification  of 
the  old  boiling  process,  as  revived  by  Mr.  Strangeways.  The 
slices  of  tissue  from  which  sections  are  to  be  cut  are  thrown  at 
once  into  boiling  water,  and  allowed  to  boil  vigorously  for  two  or 


Fig.  39. — Cathcart's  Microtome  arranged  for  cutting  Frozen  Sections. 

three  minutes  ;  the  water  must  be  actually  boiling  when  the 
tissues  are  added,  and  the  bulk  used  should  be  large  as  compared 
with  the  block.  The  tissues  are  then  rapidly  cooled  by  being 
thrown  into  cold  water,  and  are  then  ready  for  cutting.  The 
outer  surface  of  the  block  should  be  rejected. 

This  method  of  fixation  leads  to  a  little  distortion  of  the  tissues 
and  alters  any  blood  which  they  may  contain,  but  it  is  very  good 
for  diagnosing  tumours.  It  is  invaluable  in  the  post-mortem 
room,  and  for  diagnosis  of  the  nature  of  a  tumour  during  opera- 
tion. In  skilful  hands  a  section  may  be  cut,  stained,  mounted, 
and  a  diagnosis  made  in  ten  minutes  ;  or  if  no  process  of  fixation 

II — 2 


l64  CLINICAL    BACTERIOLOGY   AND    HEMATOLOGY 

by  boiling  be  used,  in  much  less.  I  have  cut,  stained,  mounted, 
and  diagnosed  a  tumour  within  three  minutes. 

A  microtome  is  necessary  for  the  successful  cutting  of  sections, 
and  the  Williams  and  Swift  patterns  are  those  in  general  use  for 
the  freezing  process.  AVe  shall  recommend  the  practitioner  who 
intends  to  take  up  this  branch  of  work  to  procure  a  Cathcart 
microtome,  which  is  exceedingly  cheap  (it  costs  about  a  guinea) 
and  answers  admirably.  The  great  advantage  of  this  machine  is 
that  it  will  serve  for  cutting  sections  in  paraffin  as  well  as  for 
frozen  sections. 

The  blocks  of  tissue  which  are  to  be  cut  are  dipped  in  a  thick 
and  syrupy  solution  of  gum  arabic ;  if  time  is  no  object  it  is  a 
great  advantage  to  soak  them  in  this  for  several  hours.  A  block 
is  then  placed  on  the  corrugated  plate  of  the  microtome  and 
frozen  by  means  of  the  ether  spray  which  impinges  upon  it. 
When  the  mass  is  nearly  frozen  a  section  is  taken  off  by  means 
of  a  razor  which  is  ground  flat  on  one  side,  or  the  special  knife 
which  may  be  obtained  with  the  apparatus ;  it  is  better  to 
moisten  the  upper  surface  of  the  knife  with  a  little  of  the  gum. 
The  section  is  carefully  removed  with  a  camel's-hair  brush  and 
placed  in  a  large  vessel  of  clean  water,  so  that  the  gum  may  be 
dissolved  out  of  it,  and  is  then  ready  for  staining.  The  block  is 
then  raised  by  means  of  a  very  slight  turn  of  the  large  milled 
head  under  the  apparatus,  and  another  section  cut. 

The  mass  must  not  be  frozen  too  hard ;  if  this  has  been  the 
case,  the  necessary  thawing  will  be  hastened  by  gently  breathing 
on  the  block.  If  it  begins  to  thaw,  a  few  squeezes  of  the  bellows 
will  bring  it  to  the  proper  consistency. 

For  extremely  rapid  work,  the  best  method  of  freezing  is  by  the 
use  of  liquid  carbon  dioxide  :  this,  however,  is  difficult  in  private 
practice,  owing  to  the  unportability  of  the  cylinders.  The  best 
method  is  to  use  ethyl  chloride  or  ansestile  in  metal  cylinders, 
such  as  are  used  for  local  anaesthesia.  The  best  method  is  as 
follows  :  Place  a  few  drops  of  water  on  the  plate  of  the  microtome, 
and  freeze  it  solid  by  the  ether  spray  whilst  the  surgeon  is 
removing  the  tumour  ;  cut  a  suitable  slice  off  the  latter  and  place 
it  on  the  layer  of  ice  thus  formed ;  direct  the  ethyl  chloride  spray 
downwards  on  to  the  slice,  which  will  be  frozen  in  a  few  seconds. 
Unless  the  water  be  previously  frozen  on  the  plate  of  the  microtome, 
the  block  of  tissue  is  very  liable  to  slip,  the  lower  portion  being 
frozen  last. 


STAINING   AND    MOUNTING    FROZEN    SECTIONS  165 

Where  very  rapid  work  is  required  it  is  not  advisable  to  stain 
the  sections  in  the  method  described  in  the  next  paragraph,  since 
it  takes  too  long.  A  simple  stain  (such  as  watery  methylene  blue) 
is  used,  the  staining  being  done  on  the  slide,  a  cover-glass  applied, 
and  the  excess  of  stain  removed  by  means  of  blotting-paper,  ft 
is  necessary  to  acquire  a  considerable  amount  of  experience  of 
this  method  before  using  it  for  diagnosis,  as  the  appearance 
of  sections  prepared  in  this  way  and  examined  in  a  watery  fluid  is 
very  different  from  that  which  they  have  when  double-stained  and 
mounted  in  balsam. 


STAINING  AND  MOUNTING  FROZEN  SECTIONS 

These  processes  are  best  carried  out  in  watch-glasses.  No 
attempt  will  be  made  to  describe  the  methods  by  which  frozen 
sections  may  be  stained  for  the  purposes  of  bacteriological  research, 
for  they  are  not  so  suitable  as  paraffin  sections  for  this  purpose. 
We  shall  describe  the  process  of  staining  in  haematoxylin  (with 
or  without  eosin  as  a  counterstain)  and  mounting  in  balsam. 

The  requisites  are :  Five  watch-glasses  containing  respectively 
haematoxylin,  watery  solution  of  eosin  (about  i  per  cent.),  alcohol 
(50  per  cent.),  absolute  alcohol,  and  clove  oil ;  a  saucer  or  other 
vessel  containing  water  to  which  a  few  drops  of  ammonia  have 
been  added ;  several  strips  of  thin  writing-paper,  each  about 
I  inch  wide  and  2  inches  long ;  some  needles,  which  may  be 
mounted  in  handles  ;  slides,  cover-glasses,  and  balsam. 

A  section  is  to  be  removed  from  the  bowl  of  water  in  which  it 
is  floating  by  means  of  one  of  the  strips  of  paper ;  this  must  be 
inserted  under  it,  and  the  section  "  pinned  "  in  place  upon  it  by  one 
of  the  needles.  A  special  section -lifter  may  be  used,  but  is  not  so 
good.  It  is  then  transferred  to  the  watch-glass  containing  the 
haematoxylin  solution,  and  the  staining  process  is  allowed  to  go 
on  for  a  minute  or  two,  a  fresh  section  being  manipulated  whilst 
it  is  taking  place.  The  first  section  is  then  removed  in  the  same 
way  as  before,  and  placed  in  the  water  containing  the  ammonia  ; 
it  soon  turns  blue,  and  when  this  is  the  case  it  is  ready  to  be 
transferred  to  the  eosin,  then  into  the  dilute  alcohol,  the  absolute 
alcohol  (where  it  should  remain  for  a  minute  or  more),  and  finally 
into  the  oil  of  cloves.  It  is  then  ready  to  be  mounted  in  balsam* 
A  convenient  way  in  which  a  section  can  be  transferred  to  a  slide 
is  as  follows :  The  section  is  carefully  spread  out  whilst  in  the 


l66  CLINICAL    BACTERIOLOGY    AND    HEMATOLOGY 

oil  of  cloves,  two  needles  being  used  for  the  purpose,  and  a  slip  of 
paper  insinuated  beneath  it.  This  strip  of  paper  is  then  drawn 
slowly  out  of  the  liquid,  and  any  folds  or  creases  which  may  be  in 
the  section  straightened  out  with  the  needles,  the  excess  of  the 
oil  of  cloves  being  allowed  to  drop  off  whilst  this  is  taking  place. 
The  strip  of  paper  is  then  inverted  (the  section  remaining  adherent 
to  the  under  surface),  placed  upon  a  clean  slide,  and  pressed  firmly 
upon  it ;  the  pressure  squeezes  out  the  greater  part  of  the  oil,  so 
that  the  section  adheres  to  the  slide,  and  the  paper  can  be  stripped 
cautiously  from  it.  A  drop  of  balsam  is  then  applied,  the  section 
covered  with  a  cover-glass,  and  examined  under  the  microscope. 

It  is  a  great  advantage  to  rinse  the  section  in  distilled  or  clear 
rain-water  after  removing  it  from  the  haematoxylin. 

The  solution  of  haematoxylin  is  best  bought  ready  made,  as  its 
preparation  is  somewhat  difficult.  Delafield's  solution  is  the  best 
for  general  work. 

A  counterstain  is  not  really  necessary  for  diagnostic  purposes, 
and  its  omission  hastens  the  process  somewhat. 

THE  PARAFFIN  PROCESS 

Tissues  which  are  to  be  cut  in  paraffin  may  be  hardened  in  any 
of  the  fluids  mentioned  above.  They  are  then  dehydrated,  cleared 
in  chloroform  or  other  fluid  which  mixes  with  alcohol  and  dissolves 
paraffin,  and  finally  soaked  in  a  mixture  of  hard  and  soft  paraffin 
kept  just  at  the  melting-point.  This  paraffin  should  be  obtained 
specially  for  the  purpose ;  the  Cambridge  paraffin  is  the  best.  It 
is  made  in  two  varieties,  the  soft,  which  melts  at  48°  C,  and  the 
hard,  which  melts  at  55°  C.  The  amounts  of  each  which  should 
be  used  for  embedding  depend  upon  the  external  temperature :  in 
very  hot  weather  hard  paraffin  may  be  used  alone,  while  under 
average  circumstances  a  mixture  of  equal  parts  of  each  is  best. 

We  shall  now  proceed  to  describe  the  various  processes  seriatim. 

Dehydration. — This  is  very  simple.  The  blocks  of  tissue  are 
placed  in  weak  spirit  for  a  few  hours  or  for  a  day,  then  changed 
into  stronger  spirit,  and  so  on  until  absolute  alcohol  is  reached. 
The  slower  this  process  is  carried  out  the  better  will  be  the  results  ; 
in  practice  the  strengths  of  the  successive  lots  of  spirit  used  may 
be  40  per  cent.,  75  per  cent.,  and  the  strong  methylated  spirit,  and 
the  block  may  remain  in  each  for  twelve  hours.  Lastly,  it  goes 
into  two  successive  lots  of  absolute  alcohol. 


THE  PARAFFIN  PROCESS  167 

In  all  cases  the  amount  of  fluid  must  be  greatly  in  excess.  It 
is  useless  merely  to  cover  the  block  with  the  spirit. 

Clarification. — In  the  next  step  the  alcohol  is  removed  from  the 
tissue  and  replaced  by  some  fluid  which  will  dissolve  paraffin. 
Fats  are  dissolved  out  from  the  tissues  at  this  stage.  — 

This  step  is  also  very  simple.  The  blocks  are  passed  directly 
from  absolute  alcohol  into  chloroform,  and  allowed  to  remain 
there  for  twelve  to  twenty-four  hours,  according  to  their  size.  It 
is  not  necessary  to  use  a  preliminary  bath  of  a  mixture  of  alcohol 
and  chloroform. 

It  is  a  good  plan  to  place  the  bottle  containing  the  block  in  a 
warm  place  with  the  cork  out  for  an  hour  or  so  before  proceeding 
further,  as  by  so  doing  the  last  traces  of  the  alcohol  will  be  removed. 

Infiltration  with  Paraffin. — This  is  the  stage  which  presents  most 
difficulties  to  the  home-worker,  for  it  is  necessary  to  keep  the 


Fig.  40. — L-sHAPED  Moulds  for  embedding  in  Paraffin. 

block  of  tissue  soaked  in  paraffin  which  is  just  melted  for  at  least 
twelve,  and  more  often  twenty-four,  hours.  To  do  this  properly 
involves  the  use  of  some  sort  of  incubator.  This  might  possibly 
be  rigged  up  out  of  a  tin  biscuit-box  in  the  manner  already 
described,  though  considerably  more  heat  would  be  necessary,  as 
the  paraffin  melts  at  about  50°  C.  But  the  writer  has  often 
embedded  the  blocks  by  placing  them  in  bottles  containing  the 
paraffin  at  such  a  distance  from  the  fire  that  the  paraffin  is  never 
completely  melted,  but  always  shows  a  thin  solid  layer  on  the 
surface.  To  do  this  it  is  only  necessary  to  look  at  the  bottle 
occasionally,  and  move  it  a  little  further  from  the  fire  if  the 
paraffin  is  completely  melted,  and  vice  versa.  The  process  may 
be  stopped  at  night  without  any  harm  resulting,  and  if  the  soaking 
only  continues  for  a  few  hours  at  a  time  it  is  of  no  consequence  so 
long  as  the  total  period  is  made  up. 

Casting  the  Blocks. — Special  metal  moulds  are  used  in  the  labora- 
tory (Fig.  40).  A  pill-box  will  do  quite  well.  A  small  amount  of 
melted  paraffin  is  poured  into  the  box,  and  the  piece  of  tissue  is 


l68  CLINICAL    BACTERIOLOGY    AND    HEMATOLOGY 

taken  from  the  bottle  containing  the  melted  paraffin  with  a  pair  of 
forceps  (previously  warmed,  so  as  to  prevent  the  paraffin  from 
setting  upon  the  points),  and  placed  in  the  paraffin  in  the  pill-box. 
It  is  necessary  to  see  that  the  surface  from  which  sections  are  to 
be  cut  should  be  placed  downwards.  The  box  is  then  filled  up 
with  melted  paraffin,  and  placed  in  a  cool  place  or  surrounded  with 
water.  The  moment  a  firm  film  has  formed  over  the  surface  the 
whole  is  plunged  in  cold  water  to  hasten  the  setting  of  the  paraffin  ; 
the  more  rapidly  this  takes  place  the  better  will  the  block  cut. 
When  the  paraffin  mass  has  hardened  completely  throughout,  it 
is  trimmed  into  shape,  taking  care  that  the  edges  of  the  surface 
which  is  to  be  cut  are  accurately  parallel. 

Cutting  the  Sections. — For  cutting  sections  in  paraffin  no 
microtome  can  be  compared  with  the  Cambridge  Rocker,  but 
very   excellent    results    can   be    obtained    by   the    use    of    the 


■ 


Fig.  41. — Clamp  for  holding  Wooden  Block  with  the  Paraffin  Block. 

Cathcart  microtome  already  mentioned.  The  paraffin  block 
containing  the  piece  of  tissue  is  mounted  on  the  freezing-plate 
of  the  microtome  (which  must  be  heated,  and  the  lower  surface 
of  the  block  pressed  upon  it),  and  the  sections  cut  in  the  manner 
described  ;  a  very  sharp  knife  is  essential,  and  the  stroke  must  be 
quicker  and  sharper  than  is  the  case  when  frozen  sections  are 
being  cut.  In  another  form  of  the  microtome  a  special  inner 
tube  is  provided  for  cutting  sections  by  the  paraffin  process. 
The  blocks  are  retained  in  place  by  a  clamp,  and  appear  in  the 
same  position  as  that  occupied  by  the  mass  of  frozen  gum.  As 
the  paraffin  is  not  sufficiently  hard  to  be  gripped  by  this  clamp, 
they  must  first  be  mounted  on  a  piece  of  wood  of  a  suitable  size 
and  shape.  This  can  be  cut  out  of  a  piece  of  firewood,  and 
should  have  one  surface  left  rough ;  this  surface  must  be  dipped 
in  melted  paraffin,  and  the  under  surface  of  the  block  partially 
melted  in  the  flame  and  pressed  firmly  upon  it.  The  piece  of 
wood  is  then  to  be  placed  in  the  jaws  of  the  clamp,  and  the  screw 
tightened  up. 


THE  PARAFFIN  PROCESS  l6g 

In  the  Cambridge  Rocker  and  in  some  other  forms  of  micro- 
tomes the  sections  adhere  to  one  another  at  the  edges,  and  form 
long  ribbons  as  they  are  cut.  In  the  Cathcart  microtome  this  is 
not  the  case,  and  each  section  must  be  dealt  with  separately  ;  it 
is  to  be  removed  carefully  from  the  knife-blade  with  a  cameFs-^ 
hair  brush  or  a  finely  pointed  pair  of  forceps,  and  placed  upon 
the  surface  of  a  bowl  of  water,  just  hot  enough  to  warm  the 
paraffin  without  melting  it.  When  this  is  done  the  sections  will 
spread  out  and  lose  all  the  creases,  and  are  then  ready  to  be 
mounted  on  slides  or  cover-glasses. 

It  often  happens  that  the  sections  roll  up  on  the  knife.  In  this 
case  they  must  be  placed  on  the  surface  of  cold  water,  and  an 
attempt  made  to  straighten  them  out  by  careful  brushing  with  a 
camel's-hair  brush ;  when  fairly  flat  they  are  to  be  lifted  up  on 
a  slide  or  piece  of  paper  (dipped  into  the  water  and  insinuated 
below  them),  and  transferred  to  the  hot  water  as  before.  But 
the  rolling  of  the  sections  may  often  be  prevented  by  sharpening 
the  knife,  by  re-embedding  the  tissues  in  harder  or  in  softer 
paraffin  according  to  the  weather,  or  by  varying  the  angle  which 
the  knife  edge  makes  with  the  glass  runners  of  the  microtome. 
These  devices  can  only  be  learnt  by  experience. 

When  the  sections  are  flattened  out  on  the  surface  of  the  hot 
water,  they  are  ready  to  be  mounted  upon  slides  or  cover-glasses : 
slides  are  by  far  the  best  for  beginners.  The  slides  (or  cover- 
glasses)  must  be  perfectly  clean,  and  are  best  kept  in  methylated 
spirit  until  they  are  to  be  used,  and  the  spirit  not  wiped  ofl". 
Each  slide  is  then  inserted  separately  into  the  water  in  an  oblique 
position,  and  the  section  moved  until  it  lies  over  the  centre  ;  the 
slide  is  then  raised  out  of  the  water,  and  carries  the  section  out 
with  it. 

The  excess  of  water  is  now  to  be  removed  by  a  piece  of  blotting 
or  filter  paper,  and  the  slide  placed  in  the  warm  incubator  for  a 
few  hours.  At  the  end  of  this  time  the  sections  will  adhere  by 
atmospheric  pressure  (like  a  boy's  leather  sucker  to  a  stone),  and 
will  not  come  off  in  the  subsequent  processes.  If  an  incubator 
is  not  at  hand  the  slides  may  be  placed  near  the  fire  (protected 
from  dust),  and  kept  at  the  body  temperature  or  a  little  higher  for 
a  few  hours  ;  the  exact  temperature  does  not  matter^  and  no  harm 
will  result  if  the  paraffin  melts,  provided  that  the  section  has 
previously  become  dry. 

In  the  older  methods  of  fixing  sections  to  the  slides  various 


170  CLINICAL    BACTERIOLOGY   AND    HEMATOLOGY 

forms  of  cements  had  to  be  used,  and  were  a  great  disadvantage. 
They  are  quite  unnecessary  except  for  sections  of  the  central 
nervous  system  ;  if  these  are  being  dealt  with  the  slide  must  be 
coated  with  a  very  thin  layer  of  a  solution  of  egg-albumin  in  water 
before  the  section  is  laid  upon  it.  The  process  is  then  exactly 
the  same  as  before. 

STAINING   AND    MOUNTING   PARAFFIN  SECTIONS 

We  will  suppose  that  the  sections  have  been  cut,  flattened  out 
on  hot  water,  and  caused  to  adhere  to  slides,  and  shall  describe 
in  general  terms  the  steps  through  which  they  must  be  taken 
before  they  are  ready  for  examination.  In  the  first  place,  it  is 
obvious  that  the  paraffin,  which  permeates  all  parts  of  the 
section  and  surrounds  it  on  all  sides,  must  be  removed,  and 
this  is  done  by  pouring  xylol,  benzine,  or  turpentine  upon  it. 
At  least  two  supplies  of  the  fluid  should  be  used,  and  it  should 
be  allowed  to  act  for  at  least  two  minutes,  the  slide  being 
rocked  all  the  time.  We  have  now  removed  the  paraffin,  and 
the  next  step  is  to  remove  the  xylol  or  other  solvent ;  this  is 
done  by  means  of  absolute  alcohol.  At  least  two  lots  should  be 
used,  and  it  should  be  allowed  to  act  for  two  minutes.  The  sHde 
is  then  washed  in  water,  and  is  ready  for  staining.  When  the 
section  is  wet  with  xylol  it  will  be  quite  transparent;  this  is 
because  the  refractive  index  of  the  xylol  is  almost  the  same  as 
that  of  glass,  and  the  rays  of  light  which  come  through  the  section 
are  not  bent.  But  when  the  alcohol  is  added  the  section  will 
suddenly  become  opaque,  and  for  the  opposite  reason. 

If  there  is  a  milkiness  on  the  section  or  slide  when  the  water 
is  poured  on,  it  is  a  sign  that  the  xylol  has  not  been  completely 
removed ;  xylol  will  not  mix  with  water,  and  forms  an  emulsion 
with  it.  If  this  should  happen,  you  must  give  the  section  another 
dose  of  absolute  alcohol,  and  rewash  in  water. 

It  is  an  advantage  to  wipe  the  surface  of  the  slide  (of  course 
avoiding  the  section)  before  going  from  one  fluid  to  another. 

A  cardinal  rule  in  dealing  with  paraffin  sections  is  never  to  let  the 
section  get  dry  from  the  moment  the  first  dose  of  xylol  is  added 
until  the  final  mounting  in  balsam. 

The  methods  of  staining  which  are  in  use  are  legion,  and  it 
would  be  far  beyond  the  scope  of  this  book  to  describe  even  a 
few  of  those  which  are  used  in  histological  work,  and  to  give 
indications  for  their  use.     It  will  be  sufficient  to  describe  (i)  a 


STAINING    AND    MOUNTING    PARAFFIN    SECTIONS  171 

method  suitable  for  the  diagnosis  of  tumours,  etc.,  and  for 
ordinary  histological  purposes  ;  (2)  a  method  of  staining  to  demon- 
strate bacteria  which  stain  by  Gram's  method  ;  (3)  a  method  for 
bacteria  which  do  not  stain  by  Gram's  method ;  and  (4)  the 
process  for  demonstrating  tubercle  bacilli  in  the  tissues.  ~ 

I.  Staining  sections  for  histological  purposes: 

1.  Xylol,  two  lots  (to  remove  paraffin). 

2.  Absolute  alcohol,  two  lots  (to  remove  xylol). 

3.  Water  (to  remove  the  alcohol). 

4.  Stain  with  haematein  (or  haematoxylin*)  for  ten  minutes  or 
more,  according  to  the  nature  of  the  specimen  and  the  con- 
dition of  the  stain.  The  exact  length  of  time  can  only  be  learnt 
by  trial,  but  ten  minutes  will  be  about  right.  Rinse  in  distilled 
water. 

5.  Wash  thoroughly  in  tap- water,  continuing  the  washing  until 
the  sections  have  a  decidedly  blue  tinge.  The  haematoxylin 
compounds  are  very  much  like  litmus,  being  red  in  presence  of 
acids  and  blue  in  presence  of  alkalies ;  the  sections  are  to  be 
coloured  blue,  and  the  necessary  alkali  is  contained  in  the  tap- 
water.  It  will  hasten  the  process  to  rinse  them  in  a  very  dilute 
solution  of  ammonia,  or,  best  of  all,  a  saturated  solution  of  lithium 
carbonate. 

6.  Stain  in  watery  eosin  for  a  minute  or  so.  This  is  the 
coitntevstain.  The  haematin  will  stain  all  nuclei  blue,  but  will 
scarcely  tinge  anything  else  ;  the  eosin  is  added  to  stain  other 
structures  a  pale  pink,  and  thus  make  them  more  visible.  It 
stains  almost  instantaneously. 

7.  Wash  off  the  eosin  under  the  tap. 

The  sections  are  now  stained.  But  they  are  opaque,  and  not 
in  a  suitable  condition  to  be  examined  under  the  microscope,  and 
are  to  be  rendered  transparent  by  being  mounted  in  balsam. 
Now,  this  cannot  be  done  in  the  same  way  as  was  used  in  the 
mounting  of  films,  for  the  drying  would  cause  the  sections  to 
shrivel  and  obscure  their  structure.  The  water  is  to  be  removed, 
it  is  true,  but  by  the  use  of  absolute  alcohol ;  at  least  two  lots 
should  be  used,  and  the  slide  rocked  from  time  to  time.  Then 
the  alcohol  (which  will  not  mix  with  balsam)  is  to  be  removed  by 
the  use  of  xylol,  balsam  added,  and  the  section  covered  with  a 
cover-glass.     The  remaining  steps  are  therefore  : 

8.  Absolute  alcohol,  two  lots  (to  dehydrate). 

*  Delafield's  haematoxylin  is  the  best  for  ordinary  work,  and  is  best  bought 
ready  made. 


172  CLINICAL    BACTERIOLOGY   AND    HEMATOLOGY 

9.  Xylol,  two  lots  (to  render  the  section  permeable  to 
balsam). 

10.  Balsam  and  a  cover-glass. 

The  last  three  steps  are  practically  the  same  as  the  first  three, 
but  in  the  reversed  order,  and  similar  phenomena  are  seen.  The 
section  is  opaque  whilst  wetted  with  the  alcohol,  and  becomes 
transparent  when  the  xylol  is  added,  and  this  transparency  is  the 
proof  that  the  steps  have  been  carried  out  properly.  If  the 
section  looks  opaque  when  held  against  a  perfectly  dark  back- 
ground, an  additional  dose  of  alcohol  must  be  used,  and  the 
xylol  applied  again. 

11.  Gram's  method  as  applied  to  sections,  suitable  for  sections 
of  diphtheritic  membrane,  organs  containing  anthrax  bacilli, 
streptococci,  staphylococci,  etc. : 

1.  Xylol,  two  lots. 

2.  Absolute  alcohol,  two  lots. 

3.  Water. 

These  steps  are  always  the  same  with  paraffin  sections,  no 
matter  what  stains  are  to  be  used  subsequently. 

4.  Aniline  gentian  violet — five  minutes. 

5.  Gram's  iodine  solution — three  minutes  or  more. 

6.  Absolute  alcohol  or  methylated  spirit — until  no  more  colour 
comes  out.  This  step  is  best  carried  out  as  follows  :  Hold  the 
slide  by  one  end,  keeping  the  fingers  clean  by  using  a  duster  or 
pair  of  dissecting  forceps,  and  pour  a  little  spirit  on  the  section  ; 
rock  it  gently  from  side  to  side  and  notice  the  clouds  of  colour 
which  it  takes  up.  After  a  little  time  pour  off  the  spirit  and  add 
a  fresh  lot  ;  repeat  the  rocking,  and  pour  off  again.  Do  this 
until  the  spirit  comes  away  quite  clean,  and  does  not  take  up  any 
colour  from  the  section.  This  may  take  a  long  or  short  time,  and 
no  definite  rules  can  be  laid  down. 

In  some  cases  decolorization  can  be  carried  out  best  by  the 
use  of  clove  oil.  This  is  applied  when  the  section  is  wet  with 
absolute  alcohol  (for  it  will  not  mix  with  water),  and  must  be 
entirely  removed  by  the  same  fluid  before  the  section  is 
mounted,  or  it  will  cause  it  to  fade.  Clove  oil  is  a  very  powerful 
decolorizing  agent,  and  requires  careful  use,  or  the  colour  may 
be  removed  from  the  bacteria. 

7.  Eosin — half  a  minute  or  more.  This  is  a  counterstain,  and 
is  used  to  demonstrate  the  structural  elements,  which  are  not 
coloured  by  the  gentian  violet.     It  may  be  omitted  in  some  cases. 


STAINING   AND    MOUNTING    PARAFFIN    SECTIONS  I73 

8.  Absolute  alcohol — two  lots  (to  remove  the  water). 

9.  Xylol — two  lots,  or  until  the  section  becomes  transparent. 

10.  Balsam  and  a  cover-glass. 

This  method  of  staining  is  very  easy  of  application,  and  the 
results  are  exceedingly  beautiful.  Bacteria  which  take  the  stain 
are  coloured  blue  or  violet,  and  actively  dividing  nuclei  and 
keratin  are  stained  in  the  same  way,  while  all  other  structures 
are  stained  pink. 

III.  Method  for  bacteria  which  do  not  stain  by  Gram's  method, 
suitable  for  sections  of  typhoid  ulcers,  lymphatic  glands  con- 
taining plague  bacilli,  etc. 

The  problem  before  us  in  this  case  is  not  at  all  easy  of  solution. 
In  the  first  place,  the  stains  which  colour  the  bacteria  also  colour 
the  tissues,  especially  the  cell  nuclei ;  the  bacteria  are  easy  to 
stain,  but  it  is  difficult  to  stain  a  section  in  which  there  is  good 
dififerentiation.  In  the  second  place,  the  stains  which  are  used  for 
bacteria  are  all  soluble  in  alcohol :  but  alcohol  is  used  to  dehydrate 
the  sections.  The  following  method  will  be  found  to  serve  fairly 
well  in  most  cases,  though  it  requires  a  certain  amount  of  practice 
for  its  successful  accomplishment. 

I,  2,  and  3.  Xylol,  alcohol,  and  water,  as  before. 

4.  Stain  in  carbol  thionin  for  ten  minutes  or  a  quarter  of  an 
hour. 

5.  Wash  in  running  water  for  ten  minutes  or  longer.  This 
removes  the  stain  from  the  tissues  before  decolorizing  the 
bacteria,  and  a  fairly  differentiated  specimen  may  be  obtained  if 
the  processes  of  staining  and  washing  are  carried  out  for  suitable 
lengths  of  time. 

Unna's  polychrome  methylene  blue  may  be  used  in  a  similar 
manner,  and  gives  even  better  results.  The  staining  should  be 
continued  for  about  ten  minutes,  and  decolorization  effected  by 
very  short  immersion  in  dilute  acetic  acid  (about  J  per  cent.), 
followed  by  a  good  washing  in  pure  water. 

6.  Remove  as  much  water  from  the  section  as  you  can  without 
actually  drying  it  by  the  cautious  use  of  clean  blotting-paper. 
Then  apply  aniUne  oil  until  the  section  becomes  perfectly  trans- 
lucent. Aniline  oil  mixes  with  water  on  the  one  hand  and  xylol 
on  the  other,  and  can  be  used  for  dehydration  just  as  alcohol 
was  ;  the  process  is  slower,  and  several  lots  of  the  oil  must  be 
used. 

7.  Wash  off  all  the  aniline  oil  by  successive  applications  of 


174  CLINICAL    BACTERIOLOGY    AND    HEMATOLOGY 

xylol.  The  permanence  of  the  preparation  will  depend  on  the 
thoroughness  with  which  this  step  is  carried  out. 

8.  Balsam  and  a  cover-glass. 

IV.  Staining  sections  to  demonstrate  the  tubercle  bacillus ; 
applicable  to  the  leprosy  bacillus  also. 

I,  2,  and  3.  Xylol,  alcohol,  and  water,  as  before. 

4.  Carbol  fuchsin  heated  until  the  steam  rises  for  five  minutes 
or  longer,  care  being  taken  that  the  section  does  not  dry  up.  Or 
the  slide  may  be  immersed  in  the  stain  and  kept  in  a  warm  place 
for  twenty-four  hours. 

5.  Dilute  sulphuric  acid  until  only  a  faint  pink  tinge  appears 
after  washing.  This  will  generally  require  an  immersion  of  ten 
minutes  or  more. 

6.  Methylene  blue  for  three  or  four  minutes.  Some  of  the 
stain  comes  out  in  the  alcohol,  so  that  the  section  must  be  stained 
more  deeply  than  will  be  required  ultimately. 

7.  Rinse  off  the  blue  stain  in  water,  and  then  remove  the  greater 
part  of  the  latter  with  blotting-paper ;  this  is  to  render  the  dehy- 
dration more  rapid. 

8.  Absolute  alcohol,  two  lots  in  rapid  succession. 

9.  Xylol. 

10.  Balsam  and  a  cover-glass. 


PART  II 

h^:matology 


ESTIMATION  OF  THE  AMOUNT  OF  HEMOGLOBIN 

The  best  form  of  haemoglobinometer  now  obtainable  is  un- 
doubtedly Haldane's  ;  it  was  introduced  since  the  first  edition 
of  this  book  (in  which  Cowers'  and  Oliver's  were  described)  was 
published,  and  is  now  generally  used.     It  is  as  simple  and  cheap 


Fig.  42.— Gowers'  H^moglobinometer. 

as  Gowers',  and  almost  as  easy  to  use,  and  gives  much  more 
accurate  results.  In  Gowers'  method  the  margin  of  error  is  very 
considerable,  though  it  will  answer  perfectly  well  when  we  simply 
wish  to  see  whether  a  patient  is  improving  under  treatment,  but 
must  not  be  relied  on  for  absolute  results.  Oliver's  haemoglo- 
binometer is  a  little  more  difficult  to  use,  but  is  somewhat  easier 
to  read ;  its  price  prevents  it  from  coming  into  universal  use. 
Gowers'  H^moglcbinometer  consists  of  two  tubes  mounted 

17s 


176  CLINICAL    BACTERIOLOGY   AND    H.EMATOLOGY 

in  a  small  stand.  One  of  these  tubes  is  filled  with  a  jelly  tinted 
to  represent  the  colour  of  normal  blood  of  a  certain  degree  of 
dilution.  The  other  is  graduated  into  a  hundred  parts,  the 
graduation  being  such  that  when  20  cubic  millimetres  of  normal 
blood  are  diluted  with  water  up  to  the  100  mark,  the  colour  of  the 
two  tubes  should  be  exactly  the  same.  A  pipette  measuring  20 
cubic  millimetres  and  a  dropping-bottle  (which  is  to  be  filled  with 
water)  are  also  provided. 

Method  of  Use. — Place  a  few  drops  of  water  (preferably,  but  not 
necessarily,  distilled)  in  the  graduated  tube.  Draw  the  blood  in 
the  usual  way.  Apply  the  tip  of  the  measuring  pipette  to  the 
drop,  and  suck  gently  until  the  blood  reaches  up  to  the  mark. 
Now  put  the  tip  of  the  pipette  into  the  small  quantity  of  water  in 
the  bottom  of  the  graduated  tube  and  blow  out  the  blood  ;  this 
will  sink  to  the  bottom  of  the  tube  ;  now  raise  the  tip  of  the 
pipette  into  the  supernatant  layer  of  clear  water;  suck  water 
up  the  pipette  until  it  reaches  above  the  mark,  and  blow  it  out ; 
repeat  this  process  until  the  blood  is  thoroughly  washed  out 
from  the  tube.  Take  great  care  not  to  withdraw  any  of  the 
diluted  blood  when  removing  the  pipette.  Finally  shake  the  tube 
so  as  to  mix  the  blood  and  water  thoroughly. 

Place  the  two  tubes  side  by  side  on  a  sheet  of  white  paper  in 
front  of  a  well-lighted  window  which  is  not  exposed  to  direct 
sunlight ;  look  at  them  by  the  light  which  is  reflected  from 
this  paper,  and  add  water  from  the  pipette  belonging  to  the 
dropping-bottle,  drop  by  drop,  until  the  colour  in  the  two  tubes 
is  exactly  the  same.  Read  off  the  height  of  the  column  of  diluted 
blood  ;  this  gives  the  percentage  amount  of  haemoglobin. 

Haldane's  H^moglobinometer  is  similar  in  principle,  but 
here  the  standard  tint  consists  of  a  sealed  tube  containing  a  solution 
of  carboxy-hsemoglobin.  The  rest  of  the  apparatus  is  exactly  like 
Gowers',  and  the  method  of  use  is  similar,  except  that,  after  the 
20  cubic  millimetres  of  blood  have  been  diluted  with  the  few  drops 
of  water  in  the  comparison-tube,  it  is  to  be  converted  into  CO  haemo- 
globin by  saturation  with  ordinary  coal-gas.  To  do  this,  take  the 
curved  tube  supplied  with  the  apparatus,  fit  it  to  an  ordinary  gas- 
burner,  and  insert  the  other  end  of  the  tube  in  the  comparison-tube, 
taking  care  not  to  touch  the  solution  of  blood  :  turn  on  the  gas,  and 
allow  it  to  run  into  the  tube  for  some  time.  When  the  comparison- 
tube  is  filled  with  gas  remove  it,  close  it  quickly  with  the  finger, 
and  shake  gently  for  a  minute  or  two  ;  if  you  wet  your  finger 


ESTIMATION    OF    THE    AMOUNT    OF    HAEMOGLOBIN         I77 

with  the  diluted  blood,  wipe  it  off  carefully  on  to  the  top,  so  as  to 
avoid  loss. 

The  remaining  steps  are  again  like  those  in  Gowers'  haemo- 
globinometer,  but  with  this  difference  :  that  you  are  comparing 
two  solutions  of  the  same  substance.  These  are  very  easy  to  match, 
and  the  exact  quality  of  the  light  does  not  matter,  so  that  the 
method  may  be  used  by  any  artificial  light. 

I  find  it  convenient  to  saturate  the  water  in  a  bottle  with 
CO  by  bubbling  coal-gas  through  it  for  some  minutes.  The 
haemoglobin  is  then  converted  into  CO  haemoglobin  in  the  process 
of  dilution,  no  further  gassing  is  necessary,  and  the  procedure  is 
exactly  like  Gowers'  in  all  respects.  The  solution  will  keep  for  a 
day  or  two  if  well  stoppered. 

Oliver's  H^moglobinometer  differs  from  that  of  Gowers  in 
that  the  degree  of  dilution  is  constant  and  the  colour  of  the  diluted 
blood  is  read  off  by  comparison  with  a  seriesof  care  fully  graduated 
standards.  It  consists  of  (i)  a  capillary  glass  tube  with  thick 
walls  and  ground  ends,  one  of  which  is  flat  and  the  other  pointed  : 
this  tube  is  mounted  in  a  metal  handle,  the  other  end  of  which 
serves  as  a  stirrer  (Fig.  43,  c)  ;  (2)  a  small  cell  with  an  opaque 
white  bottom,  and  provided  with  a  cover-glass  which  has  a  slight 
bluish  tint  {e) ;  (3)  a  series  of  twelve  coloured  glass  discs  mounted 
over  an  opaque  white  background  (a) ;  (4)  certain  small  pink  glass 
discs  used  as  riders ;  (5)  a  short  glass  pipette  with  an  indiarubber 
nipple  at  one  end  and  a  short  length  of  indiarubber  tubing  at  the 
other  (d)  :  the  latter  fits  over  the  pointed  end  of  the  capillary  tube 
mentioned  first ;  and  (6)  a  small  wax  candle  such  as  is  used  for 
Christmas-trees.  A  camera-tube  lined  with  a  green  material 
is  used  to  screen  the  eyes  whilst  the  comparison  is  being 
made. 

Method  of  Use. — Prick  the  patient  in  the  usual  way.  Apply  the 
polished  end  of  the  capillary  tube  to  the  drop  of  blood  ;  this  will 
completely  fill  the  tube,  being  drawn  up  by  capillary  attraction. 
When  quite  full,  wipe  both  ends  of  the  tube  with  the  fingers,  and 
apply  the  end  of  the  glass  pipette  (previously  filled  with  water)  to 
the  pointed  end  of  the  capillary  tube.  Now  squeeze  the  nipple 
gently,  so  as  to  force  the  blood  and  (subsequently)  the  water  drop 
by  drop  into  the  cell.  Interrupt  the  process  occasionally,  and  stir 
the  contents  of  the  cell  with  the  metal  handle  of  the  measuring- 
tube.  Continue  to  add  water  until  the  cell  is  exactly  full :  this  is 
the  first  step  which  presents  the  slightest  difficulty.     Apply  the 

12 


178 


CLINICAL    BACTERIOLOGY    AND    HEMATOLOGY 


cover-glass ;  this  must  not  enclose  any  air  under  it,  nor  cause  any 
of  the  diluted  blood  to  flow  into  the  moat  round  the  cell. 


Fig.  43.— Oliver's  H^moglobinometer. 

The  specimen  is  now  ready  for  comparison  with  the  standards. 
It  is  to  be  taken  into  a  dark  room  and  examined  by  the  light  of 


CLINICAL   APPLICATIONS  I79 

one  of  the  candles.  This  is  to  be  placed  in  front  of  the  observer 
at  a  short  distance  from  the  specimen  and  standards,  which  must 
lie  side  by  side. 

The  viewing  is  best  done  by  means  of  a  camera-tube  which 
folds  into  the  box  containing  the  whole  apparatus.  It  terminate 
in  a  diaphragm  which  is  perforated  by  two  small  holes,  one  of 
which  is  to  be  placed  over  the  centre  of  the  specimen  and  the 
other  over  the  centre  of  the  standard.  The  latter  is  to  be  moved 
about  until  a  disc  is  found  which  nearly  or  quite  corresponds  in 
colour  with  the  diluted  blood  in  the  cell.  If  the  correspondence 
is  exact,  the  process  is  at  an  end  ;  the  number  against  the  disc  in 
question  represents  the  percentage  amount  of  haemoglobin.  If 
there  is  no  disc  which  exactly  matches  the  specimen,  the  latter  is 
placed  against  the  disc  which  is  nearest  to  it,  but  not  so  deep  in 
colour.  For  example,  if  we  found  that  the  specimen  was  darker 
than  the  disc  numbered  50,  but  paler  than  that  numbered  60,  then 
it  would  be  placed  opposite  to  50.  A  slip  of  colourless  glass  is 
then  applied  over  the  specimen,  and  riders  over  the  standard  disc, 
until  an  exact  match  is  obtained.  If,  in  the  case  mentioned  above, 
we  had  to  add  a  rider  marked  5  to  the  standard  to  bring  about  an 
exact  match,  the  percentage  amount  of  haemoglobin  in  the  blood 
would  be  55. 

It  is  an  advantage  to  place  cell  and  standards  side  by  side 
rather  than  one  above  the  other,  for  the  upper  and  lower  portions  of 
the  retina  differ  in  sensitiveness  to  colour,  whilst  the  sides  do  not. 


CLINICAL  APPLICATIONS 

I.  It  is  impossible  to  estimate  even  the  presence  of  anaemia,  to 
say  nothing  of  its  degree,  without  an  examination  of  the  amount  of 
haemoglobin.  I  have  been  repeatedly  asked  by  highly  skilled 
clinical  observers  to  examine  cases  presenting  all  the  appearances 
of  anaemia  in  whom  the  blood  has  been  in  every  respect  normal. 
The  examination, therefore,  should  be  made  in  all  cases  of  supposed 
anaemia,  and  the  diagnosis  should  not  be  considered  as  established 
until  this  has  been  done. 

The  recognition  of  the  degree  of  anaemia  is  advisable,  in  that 
it  permits  the  effect  of  treatment  to  be  watched  and  ineffective 
remedies  to  be  discontinued.  It  also  affords  a  guide  as  to  prog- 
nosis, for  if  a  patient's  amount  of  haemoglobin  increases  during, 

12 — 2 


l80  CLINICAL    BACTERIOLOGY   AND    H.EMATOLOGY 

say,  the  first  week  of  treatment,  a  good  prognosis  may  be  given, 
although  there  are  no  other  signs  of  improvement,  and  some  idea 
as  to  the  time  necessary  to  effect  a  complete  cure  may  be  obtained. 

2.  Apart  from  an  ordinary  anaemia,  the  estimation  of  the  haemo- 
globin may  give  an  important  clue  as  to  the  presence  or  absence 
of  other  diseases.  For  instance,  in  severe  sepsis  there  is  usually 
a  very  marked  and  rapid  fall  in  the  amount  of  haemoglobin,  due  to 
the  destruction  of  the  red  corpuscles  by  the  toxin  of  the  infective 
organisms.  This  is  especially  valuable  in  that  it  occurs  in  severe 
infections  in  which  the  leucocytes  often  do  not  undergo  character- 
istic alterations  (p.  222).  Thus,  if  an  increasing  anaemia  is  found 
in  a  patient  during  the  puerperium,  it  points  strongly  to  puerperal 
fever,  and  the  prognosis  is  bad,  assuming,  of  course,  that  there  is 
no  haemorrhage  or  other  cause  of  anaemia. 

In  interpreting  the  results  of  this  examination  you  must  re- 
member that  when  there  is  severe  diarrhoea,  sweating,  polyuria, 
or  any  other  symptom  in  which  there  is  a  great  loss  of  water,  the 
blood  may  be  temporarily  concentrated,  and  the  amount  of  haemo- 
globin (and  of  red  corpuscles,  but  not  of  leucocytes,  or  not  to  a 
proportionate  extent)  may  appear  to  rise.  Discount  this  in  giving 
a  good  prognosis  in  septic  conditions  from  the  increase  in  the 
haemoglobin.  A  similar  concentration  may  occur  from  mitral 
disease  or  venous  stasis  from  any  cause. 

3.  A  fall  in  the  amount  of  haemoglobin  in  a  case  watched  from 
day  to  day  indicates  haemorrhage,  and  is  occasionally  valuable  in 
the  differential  diagnosis  of  internal  haemorrhage — e.g.,  in  cases  of 
ruptured  tubal  gestation. 

4.  In  malaria  there  is  a  fall  in  the  amount  of  haemoglobin,  very 
rapid  and  sudden  in  the  early  stages,  and  often  marked,  though 
less  rapid,  in  the  later  ones.  The  fall  is  often  to  an  extent  only 
equalled  in  acute  sepsis.  This  may  be  of  much  diagnostic  value. 
In  typhoid  fever  and  in  the  other  diseases  for  which  malaria  may 
be  mistaken  the  anaemia  is  usually  developed  much  more  slowly, 
if  at  all. 

5.  Considerable  help  is  afforded  in  the  diagnosis  of  syphilis  by 
Justus's  test,  which  is  based  on  the  fact  that  in  the  primary  and 
secondary  stages  of  that  disease  the  red  corpuscles  are  abnormally 
easily  destroyed  by  the  action  of  mercury,  provided  that  that  drug 
has  not  been  previously  administered.  The  test  is  also  of  value 
in  the  diagnosis  of  hereditary  syphilis. 

To  use  it,  estimate  the  haemoglobin  as  accurately  as  possible, 


ESTIMATION    OF   THE    RED    CORPUSCLES  l8l 

and  then  give  the  patient  a  single  large  inunction  of  mercury, 
thoroughly  rubbed  in :  Justus  recommends  3  grammes  (45  grains) 
for  an  adult,  i  gramme  for  an  infant.  About  twenty-four  hours 
afterwards  estimate  the  haemoglobin  again.  In  a  positive  case  there 
will  be  a  fall  of  to  to  20  per  cent.,  and  this  fall  may  continue  foF 
a  day  or  two,  and  then  give  place  to  a  gradual  rise. 

The  test  should  not  be  employed  when  there  is  marked  anaemia 
to  begin  with,  as  in  these  patients  the  blood-corpuscles  seem  very 
sensitive  to  the  mercury,  even  when  the  disease  is  not  syphilitic. 
With  these  exceptions,  I  believe  the  test  to  be  of  very  great  value, 
though  more  importance  is  to  be  attached  to  a  positive  than  to  a 
negative  result. 


ESTIMATION  OF  THE  RED  CORPUSCLES 

The  best  apparatus  for  the  estimation  of  the  number  of 
corpuscles  (whether  red  or  white)  is  the  Thoma-Zeiss  haemocyto- 
meter.  It  should  be  provided  with  two  pipettes,  one  for  counting 
the  red  corpuscles  and  one  for  the  leucocytes.  The  latter  is  rarely 
used  and  need  not  be  procured. 

Examine  the  pipettes.  Each  has  a  small  bulb  containing  a 
little  glass  ball,  and  a  stem  which  is  graduated  into  several  parts 
below  the  bulb,  and  has  a  single  transverse  graduation  above  it. 

The  pipette  intended  for  use  in  counting  the  leucocytes  may  be 
distinguished  by  the  fact  that  it  has  the  figure  1 1  over  the  single 
transverse  graduation  above  the  bulb. 

There  are  two  sorts  of  pipettes  used  for  counting  the  red 
corpuscles.  In  the  one  form  the  stem  below  the  bulb  is  divided 
into  ten  parts,  the  upper  one  (nearest  the  bulb)  being  marked  i, 
and  the  middle  one  0*5  (Fig.  44,  5).  In  the  other  one  the  same 
portion  of  the  stem  is  graduated  into  three  portions  numbered  -j-l^t 
jIq,  and  2  Jo  ;  the  figure  mentioned  first  is  placed  nearest  the  bulb. 
These  pipettes  are  used  in  the  same  way,  and  it  is  quite  immaterial 
which  is  obtained  ;  we  shall  describe  the  use  of  the  first  form. 

The  rationale  of  the  method  is  this  :  Blood  is  sucked  up  to  one 
of  the  divisions  on  the  lower  part  of  the  stem,  and  then  an  inert 
diluting  fluid  is  drawn  up  to  the  single  mark  above  the  bulb,  and 
the  two  mixed  by  rotating  the  whole  apparatus  for  a  minute  or 
two.  This  gives  us  a  dilution  of  blood  of  definite  strength,  the 
exact  amount  of  dilution  depending  upon  the  amount  of  blood 
which  was  taken.     Thus,  if  blood  had  been  drawn  up  to  the 


l82 


CLINICAL    BACTERIOLOGY   AND    HiEMATOLOGY 


figure  I,  we  should  have  a  dilution  of  i  in  loo,  while  if  blood  had 
been  drawn  up  to  the  figure  5  the  dilution  would  be  0*5  in  100, 
or  I  in  200,  and  so  on.  In  the  case  of  the  other  form  of  pipette 
the  dilution  is  read  off  directly  from  the  figures  on  the  lower  stem. 

The  diluted  blood  thus  obtained  is  spread  out  in  a  film  of  a 
definite  known  thickness  on  the  slide  supplied  on  the  instrument 
(Fig.  44,  a).  This  is  ruled  in  squares,  and  the  squares  are  of 
known  size.  The  amount  of  blood  lying  upon  each  square  is  thus 
known,  and  the  number  of  corpuscles  which  lie  upon  it  being 
counted  under  the  microscope,  all  the  data  for  the  calculation  are 
obtained. 

In  blood  examinations  it  is  absolutely  necessary  that  all  points 


C  Zeiss 
Jena. 


Fig.  44. — Thoma's  H^mocytometer, 


in  the  technique  should  receive  the  most  careful  attention,  or  the 
result  will  be  worse  than  useless.  For  this  reason  we  shall 
describe  each  step  in  the  process  at  some  length,  and  advise  the 
practitioner  to  make  several  estimations  before  placing  any 
reliance  whatever  on  his  results. 
Requisites. —  i.  The  haemocytometer. 

2.  A  needle  suitable  for  obtaining  a  small  quantity  of  blood. 
A  straight  Hagedorn's  needle  (about  2  inches  long)  is  the  very 
best  that  can  be  used,  and  an  ordinary  hare-lip  pin  will  answer 
very  well.  It  is  best  to  use  a  needle  with  aflat  cutting-point,  and 
not  a  round  or  triangular  one,  as  the  prick  is  less  painful. 

3.  Diluting  fluid.  There  are  a  good  many  formulae  for  this, 
and  some  are  rather  complicated.  Isotonic  saline  solution  (com- 
mon salt  o'8  or  thereabouts)  will  answer  perfectly  ;  it  is  advisable 
to  add  to  it  a  small  quantity  of  some  stain,  methyl  violet  being 


Estimation  oi^  the  rEd  corpuscles  183 

the  best,  although  gentian  violet  will  do  very  well.  This  colours 
the  leucocytes,  so  that  they  are  readily  distinguished  from  the  red 
corpuscles.  ■**■ 

4.  A  microscope  having  a  J-inch  lens  which  will  focus  through 
the  thick  cover-glass  supplied  with  the  haemocytometer.  If  the 
examination  is  not  to  be  made  by  the  bedside,  a  strong  indiarubber 
band  a  little  shorter  than  the  pipette  should  be  carried. 


Process. 

I.  Pricking  the  Patient. — The  blood  may  be  procured  from  the 
convex  border  of  the  lobe  of  the  ear  or  from  the  lateral  surface 
of  the  last  phalanx  of  the  finger.  The  advantage  of  the  former 
situation  is  that  the  pain  is  very  slight,  the  skin  being  thin,  and 
that  the  patient  cannot  see  what  you  are  doing,  and  is  not  likely  to 
start  at  the  critical  moment.  It  is  to  be  recommended  for  children 
and  nervous  women.  The  advantage  of  the  finger  is  that  the 
skin  is  free  from  hairs,  and  these  are  objectionable  in  the  prepara- 
tion of  films  by  the  cover-glass  method  ;  an  additional  advantage 
is  that  the  patient  can  put  his  hand  into  the  position  most  con- 
venient to  you,  and  you  have  not  to  lean  over  him. 

The  area  of  the  skin  to  be  punctured  may  be  washed  with  soap 
and  water  and  then  with  pure  water,  and  wiped  dry,  but  this  is 
not  really  necessary.  It  is  necessary,  however,  to  rub  the  patient's 
ear  or  finger  well  with  a  towel  or  piece  of  lint,  so  as  to  make  it 
hypersemic  ;  unless  you  do  this  you  may  have  difficulty  in 
collecting  sufficient  blood,  especially  if  the  skin  is  cold.  The 
needle  is  sterilized  by  being  passed  slowly  through  the  flame  of  a 
spirit-lamp  or  Bunsen  burner ;  the  area  of  skin  to  be  pricked  is 
taken  between  the  finger  and  thumb  of  the  left  hand,  and  a  rapid 
and  fairly  deep  stab  made  with  the  needle.  The  skin  is  then 
released,  and  a  drop  of  blood  allowed  to  exude  ;  this  is  wiped  away, 
and  the  next  drop  which  oozes  out  is  used  for  examination. 

The  skin  must  never  be  pinched  when  blood  is  being  with- 
drawn for  this  examination  ;  the  blood  must  always  be  allowed 
to  flow  out  naturally,  but  if  a  flat  needle  be  used,  the  edges  of  the 
cut  made  by  it  may  be  held  apart  by  gentle  pressure  with  the 
finger  and  thumb. 

*  The  following  formula  is  better :  Distilled  water,  i6o  c.c.  ;  glycerin, 
30  c.c.  ;  sodium  sulphate,  8  grammes  ;  sodium  chloride,  i  gramme  ;  methyl 
violet,  a  trace  (Toison's  fluid). 


184  CLINICAL    BACTERIOLOGY   AND    H.?iMATOLOGY 

2.  Filling  the  Pipette. — The  degree  of  dilution  is  determined  by 
the  number  of  corpuscles  per  cubic  millimetre  which  you  expect 
to  find.  If  the  patient  is  anaemic,  use  i  in  100  ;  if  he  has  approxi- 
mately the  normal  number  of  corpuscles,  or  if  you  have  reason 
to  think  that  they  are  present  in  increased  quantities,  use  a 
dilution  of  i  in  150  or  i  in  200. 

In  most  cases  you  will  find  it  advisable  to  count  the  red  and 
the  white  corpuscles  in  the  same  specimen,  and  if  this  is  the  case, 
use  a  dilution  of  i  in  100,  whether  you  expect  the  patient  to  be 
anaemic  or  not.  It  is  less  easy  to  count  the  reds  (if  numerous) 
with  this  low  degree  of  dilution  than  with  a  higher  one,  but  it  is 
not  really  difficult,  and  if  you  use  a  higher  degree  of  dilution 
considerable  error  will  be  introduced  into  the  leucocyte  count. 

Having  decided  upon  the  degree  of  dilution,  insert  the  tip  of 
the  pipette  into  the  drop  of  blood  lying  on  the  skin,  take  the  bone 
mouth-piece  attached  to  the  indiarubber  tube  in  your  mouth,  and 
suck  the  blood  up  to  the  appropriate  mark.  If  air-bubbles  gain 
access,  blow  the  blood  out  and  begin  again  quickly.  If  you  over- 
shoot the  mark,  remove  some  of  the  blood  by  touching  the  tip  of 
the  pipette  against  some  lint  or  absorbent  cotton-wool.  Be 
careful,  also,  to  wipe  off  any  blood  there  may  be  on  the  outside 
of  the  tip.  Place  the  tip  of  the  pipette  in  the  diluting  fluid  ;  a 
small  quantity  should  be  poured  out  into  a  watch-glass  or  other 
suitable  vessel,  so  as  to  avoid  any  possibility  of  allowing  some 
blood  to  escape  into  the  stock  bottle,  and  invalidating  a  sub- 
sequent observation.  Suck  the  diluting  fluid  slowly  into  the 
pipette  until  it  reaches  the  single  mark  above  the  bulb  ;  rotate  the 
pipette  between  the  finger  and  thumb  as  you  do  so. 

Now  remove  the  pipette  from  the  diluting  fluid,  place  the  tip 
of  the  finger  over  the  aperture  of  the  pipette  (Fig.  44,  S),  and 
proceed  to  mix  the  contents  by  rotating  the  pipette  and  by  turning 
it  over  and  over. 

If  the  examination  is  to  be  made  at  a  distance,  remove  the 
indiarubber  tube  and  stretch  an  indiarubber  band  over  it,  so  as 
to  close  both  apertures  of  the  pipette.  It  is  advisable  to  make  the 
examination  in  a  few  hours,  otherwise  considerable  errors  may 
creep  in. 

3.  Preparation  of  the  Specimen. — The  slide  which  is  supplied  with 
the  instrument  consists  of  a  thick  and  perfectly  flat  slip  of  glass 
(Fig.  44,  0),  on  which  is  cemented  a  glass  square  having  a  round 
hole  in  its  centre  {W).     In  the  centre  of  the  hole  thus  left  there 


\ 


ESTIMATION    OF    THE    RED    CORPUSCLES  185 

is  a  circular  disc  of  glass  (B) ;  this  inner  disc  is  made  of  glass 
which  is  exactly  y\j  millimetre  thinner  than  that  of  which  the 
outer  glass  is  constructed.  When  the  whole  cell  is  covered 
with  a  perfectly  flat  cover-glass  (D)  there  will,  therefore,  be  a 
space  exactly  y^  millimetre  between  the  lower  surface  of  this" 
cover-glass  and  the  upper  surface  of  the  central  disc ;  this  space 
is  to  be  filled  with  the  diluted  blood. 

Slide  and  cover-glass  are  to  be  wiped  clean  with  a  soft  hand- 
kerchief moistened  with  water  {not  alcohol  or  xylol,  which  may 
spoil  the  former),  and  then  thoroughly  dried  ;  there  must  not  be 
the  minutest  particle  of  dust  on  any  part  of  the  surface. 

The  slide  and  cover-glass  being  ready,  mix  the  contents  of  the 
pipette  as  you  did  before  (this  must  always  be  done  immediately 
before  making  the  specimen,  no  matter  how  carefully  it  had  been 
done  a  short  time  previously),  and  blow  out  about  half  of  the 
fluid  in  the  bulb  ;  this  is  to  wash  the  diluting  fluid  out  of  the 
lower  part  of  the  stem.  Now  clip  the  indiarubber  tube  firmly 
between  your  finger  and  thumb,  so  as  to  prevent  the  access 
of  air,  and  therefore  the  escape  of  fluid,  and  wipe  the  tip  of  the 
pipette  from  all  fluid  ;  this  may  be  done  with  the  forefinger. 
Place  the  tip  of  the  pipette  on  the  centre  of  the  central  disc  of 
the  slide,  and  relax  your  pressure  on  the  indiarubber  tube  so 
as  to  allow  a  small  drop  of  fluid  to  escape  ;  this  is  perhaps  the 
most  difficult  part  of  the  process,  and  the  exact  amount  which 
must  be  allowed  to  fall  on  to  the  slide  can  only  be  learnt  by 
experience. 

Cover  the  slide  in  this  way :  Place  your  finger  at  the  side  on 
the  glass  square  on  the  slide,  and  apply  the  cover-glass,  letting  it 
rest  against  your  finger  ;  lower  it  gently  in  place  with  a  needle  or 
other  suitable  object.  When  it  is  in  place  press  it  gently  with 
the  needle  at  each  corner  in  succession,  and  look  at  it  obliquely, 
so  as  to  see  the  light  reflected  from  the  surface.  If  the  slide  and 
cover-glass  are  in  sufficiently  close  contact,  you  will  see  Newton's 
rings  (looking  like  the  eye  of  a  peacock's  feather)  round  the  point 
at  which  you  are  applying  pressure.  If  you  do  not  see  this,  the 
inference  is  that  there  is  some  dust  between  the  slide  and  cover- 
glass  ;  you  must  clean  both  and  begin  again. 

It  is  a  great  advantage  to  clip  the  cover-glass  to  the  counting- 
chamber  until  the  corpuscles  have  had  time  to  settle.  When  this 
has  taken  place  the  depth  of  the  chamber  is  immaterial,  and  it 
does  not  matter  if  it  rises  somewhat.     The  simplest  method  is  to 


l86  CLINICAL    BACTERIOLOGY   AND    H^MATOLOGV 

use  four  Cornet's  forceps,  or  even  only  two  (applied  at  opposite 
sides),  as  in  the  figure.  Where  this  is  done  an  ordinary  No.  i 
cover-glass  may  be  used  instead  of  the  special  thick  one. 
Newton's  rings  should  appear  round  the  tips  of  the  forceps,  as 
shown  in  the  illustration.  The  preparation  should  stand  for  five 
minutes  to  allow  the  corpuscles  to  settle,  when  the  forceps  are 
removed  and  the  count  made  (see  Fig.  45). 

If  you  have  taken  the  right  amount  of  fluid,  the  drop  should 
extend  exactly  to  the  edge  of  the  central  glass  disc,  but  should 
not  run  over  into  the  "  moat  "  (Fig.  44, ;').  If  this  happens,  or  if 
there  are  any  bubbles  under  the  cover-glass,  you  must  begin  again. 


Fig.  45. — Counting  Chamber  with  Cover-glass  clipped  into  Position. 

If  the  drop  does  not  quite  extend  to  the  edge  of  the  central  disc, 
no  great  harm  is  done. 

4.  Focussing  the  Specimen. — This  is  somewhat  difficult  for  be- 
ginners, and  merits  a  short  description.  Place  the  slide  under  the 
microscope,  taking  care  to  get  it  accurately  centred,  and  examine 
it  with  the  low  power.  You  will  find  that  the  central  disc  is  ruled 
into  squares  like  a  chess-board  (Fig.  44,  c).  Get  these  squares 
into  the  centre  of  the  field  (see  Fig.  46). 

Do  not  forget  you  are  dealing  with  an  unstained  object  ;  use  a 
flat  mirror  and  a  small  diaphragm.  The  examination  is  often 
easier  if  artificial  light  is  used. 

Now  turn  on  the  high  power  (i-inch  or  i-inch),  and  screw  it 


ESTIMATION    OF   THE    RED    CORPUSCLES 


187 


downwards  until  it  almost  touches  the  cover-glass  ;  look  down  the 
microscope  and  focus  gently  upwards,  using  the  fine  adjustment, 
and  keeping  a  careful  look-out  for  the  rulings. 

Some  i-inch  lenses  focus  too  near  the  object  to  be  of  any  use. 
If  this  is  the  case,  you  must  either  get  an  objective  specially  for 
the  purpose,  or  a  cover-glass  which  is  hollowed  out  in  the  centre. 
These  can  be  bought  from  the  same  place  as  the  haemocytometer. 


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Fig.  46. — The  "Bars"  which  are  to  be  Counted  are  Shaded. 

If  the  ruUngs  of  the  slide  are  indistinct,  they  may  be  darkened 
by  rubbing  them  with  a  very  soft  lead-pencil. 

5.  Counting  the  Corpuscles. — Move  the  slide  about  until  you  have 
come  to  one  corner  (preferably  the  left  upper  corner)  of  the  ruled 
area.  You  will  see  that  each  fifth  space  is  marked  off  by  a  line 
running  down  its  centre  ;  this  is  to  guide  the  eye  and  facilitate 
counting.     The   whole  square   consists   of  four   hundred   small 


i88 


CLINICAL    BACTERIOLOGY    AND    H.EMATOLOGY 


squares,  twenty  along  each  side.  You  have  to  count  at  least  a 
hundred  of  these  small  squares.  The  simplest  way  to  do  this  is 
to  count  five  "  bars  "  of  twenty  each,  each  bar  extending  right 
across  the  ruled  "  chess-board."  The  bars  selected  should  be  as 
far  as  possible  apart  from  one  another,  so  as  to  get  a  good 
average.  In  practice  it  is  simplest  to  take  the  top  one,  and  all 
the  others  that  have  a  double  ruling ;  when  you  have  counted 
these  you  will  have  counted  five  rows  of  twenty  each  at  equal 
distances  from  one  another,  which  will  give  you  a  very  fair 
average.  (See  Fig.  46,  where  the  bars  which  are  to  be  counted 
are  shaded.) 


S'n 

a 

.JB 

t 
c 

Fig.  47. — Showing  Method  of  Counting  Red  Corpuscles. 

a,  a,  a,  are  counted  in  square  A  ;  b,  b,  inB  ;  c  in  C.     In  this  method  each 
corpuscle  is  counted  once,  and  once  only. 

In  counting  these  bars  count  all  the  corpuscles  which  are  lying 
on  or  touching  the  top  line,  as  in  the  bar  you  are  counting,  also 
those  which  are  lying  on  or  touching  the  extreme  left-hand 
limit  of  the  bar.  Exclude  those  on  or  touching  the  lower  and 
right-hand  lines.     The  reason  for  this  will  appear  subsequently. 

This  is  the  most  convenient  way  when  you  have  no  assistant 
to  take  down  numbers,  since  you  only  have  to  remove  your  eye 
from  the  microscope  for  the  purpose  of  recording  results  five 
times  in  the  whole  process,  which  takes  about  five  minutes  or 
less.  It  is,  however,  rather  more  accurate  to  take  down  the 
count  square  by  square,  dictating  the  number  to  an  assistant,  who 
tells  you  when  you  have  counted  a  hundred  ;  you  are  less  likely 


ESTIMATION    OF   THE    RED    CORPUSCLES  l8g 

to  make  errors  in  the  count  by  this  method.  If  you  follow  this 
method  in  place  of  that  given  above,  the  following  notes  should 
be  taken  into  consideration. 

If  you  exclude  the  spaces  which  are  thus  marked  with  a  double 
line,  the  whole  area  will  be  marked  out  into  a  series  of  large" 
squares,  each  consisting  of  4x4=16  smaller  squares  (Fig.  47). 
It  is  convenient  to  count  the  smaller  squares  in  these  groups  of 
sixteen.  At  least  a  hundred  of  the  smaller  squares — i.e.y  six  of 
the  large  groups  and  four  small  squares — should  be  counted. 

In  counting  one  of  the  smaller  squares  it  is  convenient  to  begin 
with  the  corpuscles  which  are  lying  in  the  middle  of  the  square, 
and  then  to  count  those  which  are  lying  on  the  lines.  In  dealing 
with  these  you  count  those  which  are  lying  on  the  tipper  and  left- 
hand  lines  as  being  within  the  square,  and  those  that  are  on  the 
lower  and  right-hand  lines  as  being  without  it ;  if  you  like  you  may 
reverse  this,  but  you  must  keep  to  the  same  method  throughout 
(see  Fig.  47). 

A  few  white  corpuscles  will  be  met  with  in  every  case,  while 
if  the  blood  was  taken  from  a  patient  with  leucocytosis  or  leuco- 
cythaemia  there  will  be  many.  They  may  be  distinguished  from 
the  red  corpuscles  by  their  greater  refractivity,  or,  if  a  stain  has 
been  used  in  the  diluting  fluid,  by  their  being  faintly  tinged.  It 
is  scarcely  necessary  to  say  that  they  should  not  be  counted. 

6.  The  Calculation. — The  best  way  of  calculating  the  number  of 
corpuscles  present  from  the  data  thus  obtained  is  the  following : 

First  add  up  the  number  of  corpuscles  in  all  the  squares  which 
you  have  counted,  and  divide  the  sum  by  the  number  of  squares 
counted.     This  gives  the  average  in  each  square. 

Now  the  space  enclosed  between  each  square  and  the 
cover-glass  above  it  is  ^^  millimetre  deep,  -gV  millimetre 
wide,  and  ^^  millimetre  long ;  its  cubic  capacity  is  therefore 
TU  ^  ^V  ^  2V  =  ToW  cubic  milUmetre.  Therefore  the  ^  ^Vcy  P^^^  of  a 
cubic  millimetre  contains  the  number  of  corpuscles  which  we 
have  already  found  as  the  average. 

But  the  square  contained  diluted  blood ;  if  the  amount  of 
dilution  was  i  in  100,  the  amount  of  blood  contained  in  the  space 
over  each  square  was  yj^^  part  of  ^xrVir  cubic  millimetre. 

Therefore  the  number  of  corpuscles  which  has  been  determined 
as  being  the  average  per  square  is  contained  in  4^V(r  ^^  twu  cubic 
millimetre  of  undiluted  blood,  the  dilution  being  taken  as  i  in  100. 

Hence   the    number   of  corpuscles  in   i   cubic   millimetre   of 


igO  CLINICAL    BACTERIOLOGY    AND    HEMATOLOGY 

undiluted  blood  is  obtained  by  multiplying  the  average  per  square 
by  the  number  which  expresses  the  dilution  (in  this  case  loo), 
and  then  by  4,000. 

It  may  be  expressed  as  a  formula,  thus  : 

If  n  is  the  total  number  of  corpuscles  counted, 

s  is  the  number  of  squares  counted, 

and  if  the  dilution  is  i  in  d, 

then     the     number     of    corpuscles     per    cubic    milli- 

metre  is  —  x  a  x  4,000. 
s 

Example. — Suppose  that  we  have  counted  100  squares,  and 
have  found  that  they  contain  1,200  corpuscles,  then  the  average 
per  square  is  12. 

Then  -^-^jj  cubic  millimetre  of  diluted  blood  contains  12  cor- 
puscles. 

Or,  Y^(y  of  tttVo  '^^^diliited  blood  contains  12  corpuscles, 
supposing  the  dilution  was  i  in  100. 

Therefore  i  cubic  millimetre  of  undiluted  blood  contains 
12  X  100  X  4,000  =  4,800,000  corpuscles. 

Or  by  the  formula — 

Number  of  corpuscles  per  cubic  millimetre  : 

1200  o 

X  4,000  X  100  =  4,000,000. 

100 

Where  the  dilution  is  i  in  100  (as  is  recommended,  since  it 
enables  the  red  corpuscles  and  the  leucocytes  to  be  counted  in  one 
specimen)  the  calculation  can  be  simplified  still  further.  Add 
up  the  number  of  corpuscles  in  the  hundred  squares  counted  and 
multiply  by  4,000.  If  you  count  200  squares,  multiply  by  2,000, 
and  so  on. 

In  a  normal  count  there  are  1,250  in  the  hundred  squares  counted ^ 
250  in  each  **  bar  "  of  twenty  squares,  and  12  J  in  each  small  square,  with 
the  dilution  of  1  in  100.  A  knowledge  of  these  facts  will  enable  the 
approximate  condition  of  the  blood  to  be  obtained  at  a  glance. 

The  beginner  is  strongly  advised  to  work  out  the  problem  at 
full  length  until  he  has  become  absolutely  familiar  with  the 
reason  for  all  the  steps. 

Clinical  Applications. 
As  this  is  more  tedious  than  the  estimation  of  the  haemoglobin, 
and  is  really  less  important  in  the  recognition  of  anaemia,  it  may 
often  be  omitted  in  clinical  work.     When  possible  it  should  be 


ESTIMATION    OF   THE    RED    CORPUSCLES  I9I 

done,  as  it  serves  as  a  useful  check  on  the  results  obtained  by  the 
estimation  of  haemoglobin.  The  normal  numbers  in  health  are 
taken  to  be  5,000,000  red  corpuscles  per  cubic  milHmetre  in  adult 
males,  and  4,500,000  in  adult  females.  As  a  matter  of  fact,  these 
figures  are  very  frequently  exceeded.  In  newly  born  children  the- 
number  is  about  5,250,000,  and  in  older  children  about  5,000,000  in 
both  sexes.    Any  decided  fall  from  these  figures  indicates  anaemia. 

The  number  of  red  corpuscles  per  cubic  millimetre  is  increased  in 
any  condition  in  which  the  total  volume  of  the  blood  is  diminished 
by  loss  of  the  fluid  portion  of  the  blood — e.g.,  in  severe  diarrhoea. 

This  fact  is  occasionally  of  diagnostic  value.  For  example,  the 
red  corpuscles  appear  more  numerous  in  typhoid  fever,  especially 
in  the  earlier  stages  of  the  disease :  the  figure  may  exceed 
6,000,000.  Later  in  the  disease  a  fall  takes  place,  though  it  is 
never  very  great,  and  if  in  a  continued  fever  the  red  corpuscles 
are  less  than  3,000,000,  the  diagnosis  of  typhoid  is  unlikely. 
This  is  very  different  to  what  happens  in  malaria,  where  there 
is  great  and  progressive  destruction  of  the  red  cells,  and  a  figure 
below  2,000,000  very  common.  This  may  be  of  value  in  diagnosis 
in  cases  where  the  parasites  cannot  be  found,  and  Widal's  reaction 
fails  or  cannot  be  tried. 

On  the  other  hand,  care  must  be  taken  not  to  mistake  this 
concentration  of  the  blood  for  an  actual  improvement — e.g.,  if  in  a 
case  of  septic  infection,  puerperal  fever,  etc.,  the  number  of  reds 
shows  a  sudden  rise,  the  question  of  whether  the  blood  has  been 
concentrated  by  profuse  diarrhoea,  sweating,  etc.,  must  be  inquired 
into  before  the  findings  raise  hope  of  a  speedy  recovery.  Mistrust 
all  results  showing  an  increase  of  more  than  100,000  red  corpuscles 
a  day.     Such  rapid  improvement  does  occur,  but  is  unusual. 

The  red  corpuscles  are  also  very  numerous  (up  to  10,000,000) 
in  congenital  cardiac  disease  with  cyanosis,  and  as  an  obscure 
primary  condition,  and  in  venous  stasis  from  mitral  disease  or 
any  other  cause. 

A  decrease  of  the  number  of  red  cells  indicates  anaemia,  and  the 
numbers  may  be  taken  as  a  criterion  of  the  degree  of  anaemia 
present.  But  this  is  not  so  accurate  a  test  as  the  percentage  of 
haemoglobin,  since  it  is  the  quantity  of  this  substance  that  is  of 
importance,  the  number  of  parcels  into  which  it  is  divided  being 
of  comparatively  little  moment.  The  grade  of  anaemia,  therefore, 
should  be  expressed  by  the  percentage  of  haemoglobin,  not  by  the 
number  of  corpuscles.     This  is  of  some  importance,  since  in  the 


192  CLINICAL    BACTERIOLOGY   AND    HEMATOLOGY 

cases  of  typhoid  fever  referred  to  above  the  haemoglobin  is  usually 
slightly  lowered,  showing  that  there  is  anaemia,  even  where  the 
number  of  corpuscles  is  abnormally  high. 

In  the  diagnosis  of  the  nature  of  an  anaemia  the  enumeration  of 
the  red  corpuscles  is  necessary,  and  the  result  is  to  be  considered 
in  connection  with  the  percentage  of  haemoglobin.  The  colour- 
index ,  or  index  of  corpuscular  richness,  is  the  figure  which  indicates  the 
richness  of  each  corpuscle  in  haemoglobin,  the  normal  figure  being 
unity.  It  is  obtained  by  dividing  the  amount  of  haemoglobin  by 
the  number  of  corpuscles,  each  being  expressed  as  a  percentage 
of  the  normal  amount.  In  health  there  are  5,000,000  red 
corpuscles  =  100  per  cent,  of  normal  and  100  per  cent,  of  haemo- 
globin.    The  index  is  therefore  : 

100 

100  ~^' 
If  we  found  a  case  in  which  the  red  corpuscles  were  4,000,000 
(  =  80  per  cent,  of  normal)  and  the  haemoglobin  40  per  cent.,  the 
colour-index  would  be  : 

40 

8o  =  °-5- 

In  another  case  we  might  find  : — Red  corpuscles  1,000,000 
(  =  20  per  cent,  of  normal),  and  haemoglobin  25  per  cent.  The 
colour-index  is  : 

-^  =  I-2S. 
20  -' 

These  figures  would  be  suggestive  of  chlorosis  and  pernicious 
anaemia  respectively. 

The  percentage  of  red  corpuscles  is  obtained  from  the  absolute 
count  by  multiplying  the  millions  figure  and  the  hundred  thousand 
figure  by  two  (since  the  normal  is  5,000,000,  the  said  figures  of 
which  become  100  when  multiplied  by  two).  Thus,  2,500,000 
=  50  per  cent. ;  900,000  =18  per  cent.,  etc.  The  same  rule  may  be 
used  for  women,  for  the  slightly  lower  normal  total  of  reds  is 
accompanied  by  a  smaller  amount  of  haemoglobin. 

The  following  are  the  general  rules  (to  which  exceptions  are 
rare)  for  the  interpretation  of  the  colour-index  : 

1.  An  index  decidedly  above  unity  indicates  pernicious  anaemia, 
and  usually  in  this  disease  the  greater  the  anaemia  the  higher  the 
index. 

2.  In  chlorosis  the  index  is  greatly  diminished  :  0*2  has  been 
recorded,  and  the  average  is  about  0*5.     The  exact  figure  throws 


ESTIMATION    OF   THE    NUMBER    OF    LEUCOCYTES 


193 


no  light  on  the  severity  of  the  case,  which  must  be  estimated  by 
the  amount  of  haemoglobin. 

3.  In  anaemia  due  to  a  single  large  haemorrhage  the  index  is  i 
at  first,  both  haemoglobin  and  corpuscles  being,  of  course,  lost  in- 
equal  proportions.  As  the  blood  begins  to  be  regenerated  it  falls 
somewhat,  not  usually  lower  than  0-9.  In  anaemia  due  to  multiple 
haemorrhages,  infectious  diseases,  poisoning,  malnutrition,  etc. 
(secondary  ancBmia),  blood  loss  and  regeneration  are  taking  place 
simultaneously,  and  the  index  falls  to  0-9,  o-8,  or  even  lower. 


ESTIMATION  OF  THE  NUMBER  OF  LEUCOCYTES 

In  clinical  work  it  is  quite  sufficiently  accurate  to  count  the  leuco- 
cytes in  the  same  preparation  as  was  used  for  the  red  corpuscles, 
and  this  is  a  great  saving  of  time  and  trouble.  Proceed  as  follows : 

Having  focussed  the  rulings  on  the  slide,  move  the  draw-tube 


^ 

^-v 

N 

/ 

N 

/ 

\ 

/ 

( 

) 

\ 

0  \ 

I 

\^ 

0 

0 

/ 

\ 

/ 

\ 

J 

\\ 

J 

\y 

Fig.  48. — Showing  Field  of  Microscope  adjusted  so  that  its  Diameter 
IS  Equal  to  that  of  Eight  Squares. 

of  the  microscope  up  and  down  until  the  upper  and  lower  limits  of 
the  field  of  the  microscope  coincide  exactly  with  two  of  the  hori- 
zontal lines,  and  count  the  number  of  spaces  (each  enclosed  between 
two  horizontal  lines)  in  the  diameter  of  the  field.  Using  a  ^-inch 
objective  it  will  be  found  possible  to  arrange  matters  so  that  these 
are  eight  in  number,  and  this  will  be  found  convenient,  though  any 
other  number  will  do.  The  essential  thing  is  that  the  upper  and  lower 
borders  of  the  field  shall  coincide  exactly  with  the  rulings.  We  will 
suppose  that  the  number  is  eight.  Then  the  diameter  of  the  field 
of  the  microscope  is  equal  to  eight  times  the  length  of  a  side  of 
a  square,  and  its  radius  is  equal  to  four  times  the  length  of  a  side 
of  a  square.     The  total  area  of  the  field  of  the  microscope  is  there- 

22  22 

fore  4  X  4  X  ~-  (ys  x  jr,  where  ir  is  taken  as  — -),  or  50  and  a  frac- 

13 


194  CLINICAL    BACTERIOLOGY   AND    HEMATOLOGY 

tion.  Practically,  therefore,  when  we  look  down  the  microscope 
after  it  has  been  adjusted  in  this  way  we  are  looking  at  fifty 
squares  ;  and  this  fact  enables  us  to  dispense  entirely  with  the 
rulings,  and  count  over  the  whole  area  of  the  disc  with  great 
rapidity.  The  slide  is  placed  in  position,  and  all  the  cells  which 
are  seen  in  the  field  counted  and  the  result  noted  down,  or, 
preferably,  dictated  to  someone  else.  The  slide  is  then  moved  on 
until  a  perfectly  fresh  portion  of  the  field  comes  into  view  ;  it  is 
advisable  to  go  too  far  rather  than  not  far  enough.  For  this 
purpose  (as  for  a  great  deal  of  blood-work)  a  mechanical  stage  is 
a  great  advantage.  In  this  way  4,000  squares — i.e.^  eighty  fields — 
may  be  counted  in  a  very  short  time. 

It  is  a  very  great  advantage  to  be  able  to  dictate  these  numbers  to 
an  assistant,  who  will  tell  you  when  forty  fields  have  been  counted. 
In  most  cases  this  will  be  enough,  but  if  the  numbers  come  out 
irregularly — ix.^  several  in  one  field  and  none  in  others — it  is  best 
to  count  eighty  fields  or  to  make  a  fresh  preparation. 

With  the  arrangement  recommended — that  is,  with  a  field  eight 
small  squares  in  diameter — you  can  tell  at  a  glance  whether  there 
is  or  is  not  leucocytosis.  //  the  leucocytes  average  one  pev  fields 
they  are  8,000  per  cubic  millimetre^  a  normal  count  ;  if  two  per 
fields  they  are  16,000  per  cubic  millimetre^  a  moderate  leucocytosis ; 
if  three  per  fields  they  are  24,000  per  cubic  millimetre,  a  high 
leucocytosis. 

The  calculation  in  this  case  is  very  simple.  If  you  have 
counted  eighty  fields,  the  total  number  is  the  number  of  leucocytes 
in  80  X  50  =  4,000  small  squares.  Now  this  is  the  number  of 
small  squares  in  a  cubic  millimetre,  so  that  the  number  only 
requires  to  be  multiplied  by  the  dilution,  in  this  case  100,  to  give 
the  number  of  leucocytes  per  cubic  millimetre.  If  you  have 
counted  eighty  squares,  therefore,  add  up  the  result  and  put  on 
two  noughts;  if  you  have  counted  forty  squares,  multiply  the 
result  by  two  and  then  put  on  two  noughts.  Thus,  if  there  are  112 
leucocytes  in  40  fields,  the  number  per  cubic  millimetre  is  22,400. 

Where  very  great  accuracy  is  required  the  special  diluting 
pipette  should  be  used.-'' 

All  the  steps  are  similar  to  those  just  described  at  full  length, 
except  that  a  different  diluting  fluid  is  used. 

*  I  allow  this  statement  to  stand,  but  have  now  great  doubts  as  to  its 
corre2tness,  and  believe  that  the  results  obtained  by  the  field  method  are 
even  more  accurate,  especially  in  unpractised  hainds, 


ESTIMATION    OF   THE    NUMBER    OF    LEUCOCYTES        ig5 

The  diluting  fluid  is  one  which  destroys  ("lakes")  the  red 
corpuscles,  but  does  not  injure  the  leucocytes.  It  consists  of  a 
0-3  or  0*5  solution  of  acetic  acid  (glacial)  in  water ;  it  is  better  to 
add  a  small  quantity  of  methyl  violet  or  gentian  violet,  so  that  the_ 
leucocytes  are  stained  and  thereby  rendered  more  prominent. 
This  solution  is  best  prepared  fresh,  or  at  any  rate  kept  in  a  well- 
stoppered  bottle. 

The  pipette  is  distinguished  from  that  used  for  the  red  corpuscles 
by  its  having  the  number  1 1  above  the  bulb.  This  indicates  that 
if  blood  be  sucked  up  to  the  mark  i  below  the  bulb,  and  diluting 
fluid  up  to  the  transverse  mark  above  the  bulb,  the  dilution  will 
be  I  in  10,  and  so  on. 

The  blood  should  be  sucked  up  to  the  mark  i  if  a  great  excess 
of  leucocytes  is  not  expected.  If  the  case  is  one  of  leucocytosis, 
a  greater  dilution  is  better  ;  whilst  if  there  is  a  great  excess  of 
leucocytes  (such  as  occurs  in  severe  leucocythaemia),  it  is  best  to 
use  the  red  corpuscles  pipette  with  a  dilution  of  i  in  100,  but 
employing  the  acetic  acid  diluting  fluid.  Then  proceed  to  make 
the  preparation,  and  count  by  the  field  method  as  above. 

Immediately  after  use  the  pipettes  must  be  thoroughly  cleaned. 
The  fluid  which  remains  in  the  bulb  must  be  blown  out,  and  for 
this  purpose,  as  well  as  for  the  subsequent  washings,  it  is  an 
advantage  to  reverse  the  position  of  the  indiarubber  tube,  so  that 
the  fluid  may  be  blown  out  through  the  upper  part  of  the  pipette, 
this  being  the  wider.  The  whole  pipette  must  now  be  filled  with 
water  (preferably  distilled),  and  the  water  blown  out.  This  process 
is  repeated,  using  absolute  alcohol,  and  allowing  it  to  run  out  of 
the  pipette  without  blowing  it.  Lastly,  fill  the  whole  pipette  with 
ether,  remove  the  indiarubber  tube,  replace  it  with  the  tube  of  an 
ordinary  spray  (such  as  is  used  for  scent  fountains,  throat  sprays, 
etc.),  and  pump  air  through  until  the  apparatus  is  absolutely  dry. 
You  can  tell  when  this  has  happened  by  the  fact  that  the  ball 
inside  the  bulb  will  emit  a  clear  ringing  sound  when  the  pipette 
is  shaken.  It  is  useless  to  attempt  to  dry  the  tube  by  blowing 
through  it  from  the  mouth. 

If  blood  has  coagulated  within  the  apparatus,  it  must  be  digested 
out.  Fill  the  whole  with  an  artificial  digestion  fluid  (pepsin  and 
very  dilute  hydrochloric  acid),  and  place  it  in  a  test-tube  of  the 
same  fluid  in  a  warm  place  for  twenty-four  hours.  Then  try  to 
clean  it  as  before,  and  repeat  the  digestion  if  this  is  impossible. 

13 — 2 


196  CLINICAL    BACTERIOLOGY   AND    HEMATOLOGY 

Clinical  Applications. 

The  clinical  applications  of  the  leucocyte  count  are  so  wide 
that  it  is  hardly  possible  to  summarize  them  here  ;  it  is  more 
convenient  to  refer  to  each  special  case  separately  under  the 
heading  of  the  blood-count  as  a  whole.  For  example,  in  dealing 
with  typhoid  fever  the  leucocyte  count  is  explained  and  the  points 
on  which  a  diagnosis  is  framed  are  given,  which,  in  a  case  in 
which  there  is  a  doubt  as  to  the  diagnosis  between  this  con- 
dition and  pneumonia,  may  be  referred  to  in  conjunction  with  the 
account  of  the  latter. 

It  will  be  convenient,  however,  to  give  a  list  of  the  usual 
counts  met  with  in  certain  diseases,  classified  under  five  headings 
in  respect  of  the  number  of  leucocytes  to  be  expected  in  them. 
But  these  figures  must  always  be  considered  with  reference  to 
the  fuller  accounts  of  the  diseases  in  question. 

I.  Enormous  Leucocytosis  (100,000  to  1,000,000). — Such 
figures  are  practically  only  met  with  in  myelogenous  or  lymphatic 
leucocythaemia,  though  suppuration,  pneumonia,  and  hooping- 
cough  may  very  rarely  approximate  thereto. 

II.  High  Leucocytosis  (20,000  to  100,000). — Suppuration  in 
all  situations  and  of  all  kinds,  the  degree  of  the  leucocytosis  being 
a  measure  of  the  virulence  of  the  organism  and  the  resisting 
power  of  the  patient. 

Pneumonia,  in  which  the  same  facts  hold. 

Hooping-cough. 

Meningitis,  especially  suppurative  meningitis,  whether  cerebro- 
spinal, pneumococcic,  etc.  In  tuberculous  meningitis  there  may 
be  as  many  as  25,000  leucocytes. 

Pleurisy,  especially  empyema.  A  leucocyte  count  of  20,000 
does  not  exclude  tuberculous  pleurisy,  but  tells  strongly  against  it. 

Scarlet  fever. 

Diphtheria. 

Hemorrhage. — After  a  very  severe  haemorrhage,  whether  internal 
{e.g.,  rupture,  extra-uterine  gestation)  or  external  {e.g.,  from 
gastric  ulcer),  figures  over  20,000  are  occasionally  seen,  but 
usually  rule  lower  (12,000  to  15,000).  The  number  usually  falls 
in  a  few  days. 

Similar  figures  are  seen  immediately  before  death,  especially  if 
the  death  is  lingering,  and  very  high  figures  are  occasionally  seen. 
It  is  sometimes  of  value  in  prognosis. 


ESTIMATION    OF  THE    NUMBER   OF   LEUCOCYTES  I97 

In  ovarian  cysts  with  twisted  pedicle,  in  intussusception  and 
volvulus,  the  figures  may  exceed  20,000. 

III.  Moderate  Leucocytosis  (10,000  to  20,000). — Here  come 
mild  cases  of  almost  all  the  diseases  mentioned  above.  Thus,  a 
very  mild  case  of  pneumonia  may  show  a  leucocytosis  within 
these  limits,  or  be  normal. 

Inflammatory. — Leucocytosis  in  inflammatory  conditions  other 
than  suppuration  rarely  exceeds  20,000,  and  does  not  often  reach 
16,000  :  this  is  a  most  important  diagnostic  feature. 

Rheumatism,  in  the  absence  of  complications. 

Tonsillitis,  except  when  very  severe,  when  higher  figures  may 
be  seen.  • 

Secondary  anamia  (see  p.  217). 

Gout. — Here  the  condition  is  normal  during  the  interval,  with  a 
slight  leucocytosis  during  the  attack. 

Small-pox,  during  the  pustular  stage.  In  severe  cases  the  count 
may  exceed  20,000,  and  figures  much  above  this  indicate  a  bad 
prognosis. 

Perforation  in  Typhoid  Fever. — Here  the  leucocytosis  usually 
attains  15,000  in  less  than  an  hour,  and  may  go  much  higher. 

Secondary  syphilis  in  most  cases. 

Malignant  Tumours. — There  is  often,  but  not  invariably,  a  slight 
rise  in  the  leucocytes  with  malignant  tumours,  but  it  is  hardly 
marked  in  the  early  stages. 

In  addition  to  these  pathological  leucocytoses,  there  are  two 
physiological  conditions  associated  with  a  moderate  leuco- 
cytosis. 

Digestion. — Under  normal  conditions  there  is  a  rise  of  i  ,000  to 
3,000  soon  after  a  meal.  This  must  always  be  remembered  in 
interpreting  a  leucocyte  count.  A  figure  of  12,000  obtained  in 
a  patient  an  hour  or  two  after  a  full  meal  does  not  neces- 
sarily indicate  disease.  If  possible,  examine  the  patient  when 
fasting. 

Pregnancy. — The  figures  rise  gradually  toward  the  end  of 
pregnancy,  and  at  term  may  reach  15,000  or  even  higher. 
Recollect  this  in  interpreting  counts  in  pregnancy  or  soon  after 
parturition. 

IV,  Normal  Counts  (5,000  to  10,000,  average  7,500)  are 
met  with  in  a  variety  of  conditions,  but  in  comparatively  few 
that  are  attended  with  pyrexia.  Of  these  the  most  important 
are : 


198  CLINICAL    BACTERIOLOGY    AND    HEMATOLOGY 

Tuberculosis,  except  tuberculous  meningitis  (15,000  to  25,000), 
and  occasionally  in  tuberculous  pleurisy  (10,000  to  20,000). 

Typhoid  fever. 

Malta  fever. 

Measles. 

Malaria. 

Mumps. 

Varicella. 

Pernicious  ancsmia. 

Chlorosis. 

Primary  syphilis. 

Influenza.  • 

In  any  of  these  there  will  be  leucocytosis  if  an  inflammatory 
complication  is  present.  Thus,  in  typhoid  fever  with  perforation 
or  pneumonia  the  count  is  raised. 

Very  severe  sepsis. 

V.  A  Lowered  Count,  or  Leucopenia  (under  5,000). — This 
is  not  met  with  very  frequently,  and  all  the  cases  are  included 
under  IV.  The  chief  are  typhoid  fever,  pernicious  anamia,  chlorosis, 
influenza,  malaria,  and  uncomplicated  tuberculosis.  It  is  also  found  in 
starvation  and  malnutrition. 


THE   INVESTIGATION    OF   THE   MORPHOLOGY  OF 
THE  LEUCOCYTES  AND  RED  CORPUSCLES 

To  study  the  morphology  of  the  leucocytes  and  red  corpuscles 
it  is  necessary  to  prepare  thin  and  even  films  of  the  blood,  and  to 
submit  them  to  appropriate  methods  of  staining. 

Method  of  Preparing  Films — I.  With  Cover-glasses. 

This  is  the  best  method  for  ordinary  purposes,  and,  if  the  in- 
structions are  carried  out  exactly,  is  a  very  easy  one. 

Requisites. — i.  Perfectly  clean  cover-glasses.  They  should  be 
cleaned  by  one  of  the  methods  described  on  p.  31,  and  kept  in 
spirit.  Immediately  before  they  are  required  for  use  they  must 
be  removed  with  a  clean  pair  of  forceps  and  dried  with  an  old 
and  soft  handkerchief.  They  may  then  be  passed  rapidly  through 
the  flame,  and  allowed  to  cool. 

I  prefer  No.  2  cover-glasses  for  this  examination,  as  much 
better  films  are  made  on  them  than  on  thin  ones,  which  are 


MORPHOLOGY   OF    LEUCOCYTES    AND    RED    CORPUSCLES    IQQ 

liable  to  bend  under  the  powerful  suction  exerted  by  the  capillary 
attraction  of  the  drop  of  blood  when  spread  out  thin. 

2.  A  needle  for  drawing  blood. 

(3.  A  platinum  loop  having  a  diameter  of  about  y^^j  inch  is 
sometimes  useful,  especially  to  a  beginner.) 

Rub  the  patient's  ear  or  finger  thoroughly  with  a  piece  of  lint  or 
a  towel,  so  as  to  make  it  hyperaemic.  Prick  it,  and  wipe  away 
the  first  drop  of  blood.  Then  allow  another  drop  to  exude  ;  if 
necessary,  you  may  squeeze  it  a  little. 

Take  a  cover-glass  between  the  first  finger  and  thumb  of  the 
left  hand,  holding  them  by  the  opposite  angles,  and  take  another 
between  the  first  finger  and  thumb  of  the  right  hand,  holding  it 
by  adjacent  angles  (Fig.  49). 


Fig.  49. 

Touch  with  the  upper  surface  of  the  cover-glass  in  your  left 
hand  the  drop  of  blood  on  the  patient's  skin,  so  as  to  remove  a 
very  small  droplet.  This  is  the  most  difficult  step  :  you  must 
not  get  too  much  or  too  little  blood,  otherwise  the  films  will  be 
useless.  It  is  advisable  to  avoid  letting  the  cover-glass  touch  the 
patient's  skin. 

Now  put  the  right-hand  cover-glass  over  the  left-hand  one,  the 
centres  coinciding  ;  lower  the  upper  (right-hand)  one  until  the 
droplet  of  blood  just  touches  it,  and  then  let  go  (Fig.  50). 

You  will  see  the  droplet  of  blood  spread  itself  out  by  capillary 
attraction  between  the  two  cover-glasses. 

At  this  stage  you  will  see  whether  you  have  taken  the  right 
amount  of  blood  or  no.  If  you  have,  the  drop  will  spread  out, 
still  retaining  its  circular  shape,  until  it  approaches  the  octagon 
formed  by  the  intersecting  edges  of  the  two  cover-glasses 
(Fig.  50) ;  if  you  have  taken  too  little  it  will  not  reach  so  far, 
and  if  you  have  taken  too  much  it  will  extend  further,  and  the 
upper  cover-glass  will  float  loosely  on  the  lower. 


200 


CLINICAL    BACTERIOLOGY   AND    HEMATOLOGY 


It  is  necessary  to  lay  great  emphasis  on  the  fact  that  the  cover- 
glasses  must  not  be  squeezed  together,  but  must  simply  come 
together  by  capillary  attraction. 

When  the  drop  has  ceased  to  spread  take  hold  of  the  upper 
cover-glass  with  the  finger  and  thumb  of  your  right  hand  and 
slide  the  two  apart,  keeping  them  in  the  same  plane;   this  is 


Fig.  50. 

readily  done,  since  the  cover- glasses  are  free  to  turn,  being  only 
held  loosely  at  the  points.  If  you  do  not  do  this,  either  the  cover- 
glasses  will  break,  or  else  the  upper  cover-glass  be  lifted  from  the 
lower  one,  and  the  film  will  resemble  the  marks  left  on  a  knife  which 
has  been  pressed  on  butter  and  lifted  off;  such  films  are  useless. 
Here,  again,  you  find  whether  you  have  taken  the  right  amount 


Fig.  51. 

of  blood.  If  you  have  taken  too  little,  the  cover-glasses  will  be 
very  difficult  to  separate ;  it  may,  indeed,  be  impossible  to  do  so 
without  breaking  them.  If  you  have  taken  too  much,  they  will 
separate  with  great  readiness,  and  the  blood  will  spread  in  uneven 
smears  instead  of  forming  a  uniform  film. 

I  used  formerly  to  recommend  the  use  of  forceps  for  holding 
the  cover-glasses  in  making  these  films.  The  only  advantage  is 
that  it  avoids  "  steaming  "  the  cover-glasses  by  the  condensation 


MORPHOLOGY  OF  LEUCOCYTES  AND  RED  CORPUSCLES  20I 

of  moist  are  from  the  fingers,  which  may  distort  the  corpuscles. 
Except  when  the  operator's  fingers  are  very  moist  it  is  quite 
unnecessary,  and  perfect  films  may  be  obtained  with  the  cover- 
glasses  held  in  the  fingers. 

The  blood  may  be  taken  by  means  of  the  platinum  loop,  and' 
this  is  a  good  plan,  as  all  danger  of  smearing  the  cover-glass 
upon  the  skin  is  avoided.  If  several  films  are  to  be  taken,  a 
number  of  platinum  loops  should  be  provided,  as  the  blood  upon 
them  soon  coagulates.  The  exact  size  of  the  loop  can  only  be 
learnt  by  experiment,  and  when  one  has  been  found  to  deliver 
a  drop  of  the  right  size  it  should  be  kept  entirely  for  this  work 
and  carefully  protected  from  injury. 


Fig.  52. — Method  of  spreading  Films  with  Cigarette-paper. 


II.  Method  with  Cigarette-papers. 

Requisites. — i.  Perfectly  clean  slides. 

2.  Some  fairly  stiff  cigarette-papers  cut  in  half  longitudinally. 
Paper  which  is  decidedly  ridged  or  ribbed  will  not  answer. 

3.  Needle. 

Method. — The  patient  is  pricked,  and  the  first  drop  of  blood 
wiped  away  as  before.  One  of  the  half  strips  of  cigarette-paper 
is  now  held  in  the  right  hand,  the  index-finger  being  placed 
above  the  strip,  and  the  edges  held  between  the  thumb  and 
index-finger  and  the  index  and  middle  fingers  respectively ;  this 
converts  it  into  a  gutter,  the  convex  edge  of  which  is  downward. 
The  edge  of  this  gutter  which  points  away  from  you  (and  which 
is  formed  by  a  machine-cut  edge  of  the  paper)  is  now  dipped  into 
the  drop  of  blood,  and  a  small  quantity  picked  up  on  its  lower 
surface.  This  lower  surface  is  then  placed  on  a  clean  slide 
parallel  to  one  of  its  shorter  edges  and  about  J  inch  from  it,  and 
pressed  gently  upon  it  so  as  to  flatten  out  the  paper  gutter  ;  as 
this  flattens  out  the  edge  of  the  drop  of  blood  on  its  under 
surface  will  follow  it.     The  strip  of  paper  is  now  drawn  towards 


202  CLINICAL    BACTERIOLOGY   AND    HEMATOLOGY 

the  other  end  of  the  sUde  with  a  steady  uniform  movement,  and 
in  doing  so  the  drop  of  blood  is  spread  out  into  a  long  uniform 
film.  In  this  way  a  film  J  inch  wide  and  2  inches  long  can  be 
made  on  a  single  slide.  A  fresh  piece  of  paper  is  to  be  used  for 
each  specimen  (see  Fig.  52). 

The  author  is  of  opinion  that  it  is  best  to  adopt  the  cover-glass 
method,  as  he  has  found  that  it  presents  fewer  difficulties  for 
beginners ;  this  is  not  the  universal  experience,  and  it  is  a  good 
plan  to  try  both,  and  adopt  that  with  which  you  get  the  best 
results. 

Films  are  sometimes  spread  on  one  slide  by  means  of  another, 
which  is  used  as  a  spreader  in  much  the  same  way  as  the 
cigarette-paper  described  above.  This  is  very  good  for  malaria 
parasites,  and  for  alterations  in  the  red  corpuscles,  but  is  useless 
for  making  differential  counts,  as  some  of  the  leucocytes  are 
carried  along  with  the  spreader  and  left  at  the  end  of  the  film. 

Fixation  of  Blood-films. 

If  films  are  required  simply  for  bacteriological  purposes  {i.e.,  to 
search  them  for  bacteria),  they  may  be  fixed  by  passing  them 
three  times  through  the  flame,  just  as  if  they  were  ordinary  films. 
This,  however,  is  not  to  be  recommended  in  the  study  of  the 
cells  of  the  blood  or  of  the  parasite  of  malaria,  though  it  answers 
very  well  in  skilful  hands.  Three  methods  should  be  learnt :  the 
method  by  heat,  the  alcohol  and  ether  method,  and  the  formalin 
method.  Of  these,  the  first  is  necessary  if  Ehrlich's  triacid  stain 
is  to  be  used,  but  the  others  are  perhaps  better  for  other  stains. 
In  addition  to  these  we  must  mention  that  if  Jenney's  stain  is  used 
a  preliminary  fixation  is  unnecessary,  as  the  fluid  fixes  the  film  and 
stains  it  at  the  same  time.  This  is  the  method  of  blood  examination 
which  is  most  suitable  for  practitioners,  and  it  is  doubtful  whether 
it  is  not  also  the  best  for  the  most  accurate  and  delicate  scientific 
research. 

I.  Method  of  Fixation  by  Heat. — Slides  or  cover-glasses  to  be 
fixed  by  this  method  must  be  exposed  to  a  temperature  of  120°  C. 
for  about  five  minutes — slides  requiring  a  slightly  longer  time 
than  cover-glasses. 

The  ideal  way  is  to  use  a  dry-air  sterilizer  (see  p.  5),  to  place 
the  films  in  it  whilst  cold,  to  heat  up  to  120°  C,  and  then  to  turn 
out  the  gas.  In  the  absence  of  this  apparatus  a  metal  slab  or 
plate  such  as   has   been  recommended   for   use  in  staining  the 


FIXATION    OF    BLOOD-FILMS  203 

tubercle  bacillus  answers  well.  It  is  mounted  upon  a  tripod 
stand,  and  the  heat  applied  at  one  end.  After  a  time  the  tem- 
perature of  various  portions  of  the  plate  is  tested  by  the 
application  of  a  few  drops  of  water  ;  the  point  at  which  the  drop 
assumes  the  "spheroidal  state"  {i.e.,  takes  the  form  of  a  sphere, ~ 
and  does  not  wet  the  plate)  is  about  the  right  point  to  use.  The 
slides  or  films  are  placed  at  this  point  for  the  appropriate  time. 

II.  Fixation  by  Perchloride  of  Mercury. — Flood  the  film  with  a 
saturated  watery  solution  of  perchloride  of  mercury,  allow  to  act 
for  a  minute,  and  wash  for  a  minute  under  the  tap,  or  by  washing 
it  in  a  vessel  of  water. 

This  method  of  fixation  is  about  the  best  that  can  be  used  for 
general  purposes. 

III.  Fixation  by  Alcohol  and  Ether. — This  is  very  simple  ;  the 
films  are  placed  in  a  mixture  of  equal  parts  of  alcohol  and  ether 
for  at  least  half  an  hour. 

This  method  of  fixation  is  good,  but  slow. 

IV.  Fixation  by  Formalin. — There  are  several  methods  by  which 
the  fixative  action  of  formalin  can  be  used  for  blood-work.  Of 
these,  the  use  of  a  mixture  of  i  part  of  formalin  with  9  parts 
of  absolute  alcohol  answers  perfectly.  The  films  are  immersed  in 
this  for  half  a  minute,  and  then  washed  thoroughly  under  the  tap. 

This  method  of  fixation  is  both  good  and  rapid. 

Staining  Blood-films  for  the  Investigation  of  their  Cells. 

There  are  a  great  many  methods  of  staining  blood-films,  and 
all  depend  upon  the  division  of  stains  into  two  varieties,  the  acid 
and  the  basic.  All  the  stains  which  are  used  in  this  branch  of 
histology  are  salts,  and  in  some  of  these  salts  the  acid  radicle 
does  the  staining,  in  others  the  basic. 

Acid  stains  are  those  in  which  the  colouring  property  resides  in 
the  acid  of  the  salt.  A  familiar  example  is  picrate  of  potash,  a 
yellow  stain  in  which  the  picric  acid  is  the  active  ingredient. 
The  acid  stains  in  chief  use  are  eosin,  acid  fuchsin,  and  orange 
G.  Substances  which  stain  with  an  acid  stain  after  suitable 
exposure  to  a  mixture  of  an  acid  and  a  basic  stain  are  called 
oxyphile,  or,  from  the  frequent  use  made  of  eosin  as  an  acid  stain, 
eosinophile. 

Basic  stains  are  those  in  which  the  colouring  property  resides 
in  the  basic  radicle  of  the  salt ;  they  include  all  the  stains  which 
are  in  use  for  staining  bacteria,  and  they  all  colour  the  nuclei  of 


204  CLINICAL    BACTERIOLOGY   AND    HEMATOLOGY 

cells.  The  most  important  are  methylene  blue,  methyl  green, 
and  toluidin.  Ordinary  basic  fuchsin  used  in  staining  the  tubercle 
bacillus  belongs  to  this  group,  as  do  haematoxylin,  carmine,  etc. 

We  shall  describe  three  methods  of  staining,  and  these  are 
sufficient  for  all  purposes  of  diagnosis.  They  are :  (i)  Ehrlich's 
method  with  his  triacid  stain ;  (2)  Jenner's  method ;  and 
(3)  eosin  and  methylene  used  separately.  Of  these,  the  second 
method  is  the  simplest,  and  all  that  is  necessary  in  the  vast 
majority  of  cases.  The  third  method  is  an  emergency  one,  for 
use  when  Jenner's  stain  is  not  at  hand :  the  first  is  now  almost 
obsolete,  but  occasionally  useful  when  a  study  of  the  granulations 
of  the  leucocytes  is  of  importance. 

1.  Ehrlich's  stain  consists  of  a  mixture  of  acid  fuchsin,  orange 
G,  and  methyl  green  dissolved  in  water,  glycerin,  and  alcohol. 
It  is  difficult  to  prepare,  and  should  be  purchased  from  a  trust- 
worthy maker.  Its  use  is  very  simple.  The  film  is  fixed  by 
heat  in  the  manner  already  described,  and  the  stain  is  poured  on 
to  it,  and  allowed  to  act  for  five  minutes.  The  film  is  then 
washed,  dried  with  blotting-paper,  and  then  by  gentle  heat,  and 
mounted  in  balsam. 

Nuclei  are  stained  green,  red  blood-corpuscles  orange,  and 
eosinophile  granulations  bright  red.  The  small  eosinophile 
granulations  which  are  present  in  the  polymorphonuclear  cells 
(the  neutrophile  granulations  of  Ehrlich)  are  stained  a  purplish  or 
coppery  colour.     The  basophile  granulations  are  unstained. 

This  stain  is  not  suitable  for  the  parasite  of  malaria,  nor  for 
bacteria. 

2.  Jenner's  stain  consists  of  a  solution  of  a  compound  of  eosin 
and  methylene  blue  in  methyl  alcohol.  It  must  be  bought 
ready  prepared.  Nothing  could  be  more  simple  than  the  way  in 
which  it  is  used ;  no  preliminary  fixation  is  necessary,  the  film 
being  allowed  to  dry  and  flooded  with  the  stain.  After  a  period 
of  from  a  minute  and  a  half  to  three  minutes,  the  stain  is  washed 
off  by  waving  the  film  to  and  fro  in  distilled  or  rain  water  for  a 
few  seconds,  and  the  specimen  dried  by  blotting  it  between  two 
pieces  of  clean  blotting  or  filter  paper,  allowed  to  get  quite  dry, 
and  mounted  in  balsam  or  cedar-oil,  and  the  specimen  dried  and 
mounted.* 

*  It  has  been  objected  that  Jenner's  method  does  not  always  give  good 
results,  and  that  the  above  is  an  insufficient  account  of  the  process.  Provided 
that  the  stain  is  good  (Grubler's  can  always  be  relied  on,  and  will  keep  for 


STAINING    BLOOD-FILMS  205 

After  the  use  of  this  stain  nuclei  are  stained  blue,  red  corpuscles 
red,  eosinophile  granules  red,  and  basophile  granules  violet.  The 
descriptions  of  the  leucocytes  and  abnormal  red  forms,  which  are 
appended,  are  all  based  on  the  appearances  seen  in  films  stained 
by  this  method,  from  which  also  Plates  VII.  and  VIII.  were  drawn. 

Jenner's  stain  is  suitable  for  a  study  of  the  parasite  of  malaria, 
which  it  stains  blue.    It  may  be  used  for  the  detection  of  bacteria. 

Leishmans  stain  is  similar  in  some  respects  to  Jenner's,  and  is 
used  as  follows :  The  film  is  flooded  with  the  stain,  which  is  allowed 
to  act  for  two  minutes  ;  two  or  three  drops  of  distilled  water  are 
then  added,  and  the  process  allowed  to  continue  for  two  or  three 
minutes  longer.  It  is  then  washed,  dried,  and  mounted  as  above. 
The  colours  of  films  stained  by  this  method  differ  from  those  in 
which  Jenner  has  been  used,  the  main  point  being  that  the  nucleus 
of  the  leucocytes  is  a  fine  purplish  red  ;  the  leucocytes,  however, 
can  be  easily  recognised  from  the  coloured  plates. 

Leishman's  method  is  the  best  for  working  with  malaria  and  all 
parasitic  protozoa,  the  nuclei  of  which  are  stained  a  bright  red. 
For  ordinary  blood-work  I  personally  prefer  Jenner  (perhaps 
because  I  am  more  used  to  it),  and  the  practitioner  is  recommended 
to  choose  one  process  and  stick  to  it.  If  he  is  likely  to  need  it  for 
malaria  or  other  parasites,  Leishman's  stain  should  be  used. 

3.  Eosin  and  methylene  blue  used  separately. 

In  this  method  the  films  are  to  be  stained  with  the  eosin  first, 
and  then  with  the  methylene  blue.  Its  successful  application 
requires  a  certain  amount  of  practice. 

The  eosin  used  must  be  in  watery  solution,  and  the  exact 
strength  does  not  matter  :  4  per  cent,  is  a  convenient  strength  to 
use.  Most  specimens  of  red  ink  (slightly  diluted)  will  do  quite 
well.  The  films  are  to  be  stained  in  this  solution  for  three  or 
four  minutes  ;  no  harm  will  result  if  they  are  left  in  much  longer. 
They  are  then  washed  and  immersed  in  a  saturated  watery 
solution  of  methylene  blue.  This  is  the  difficult  part  of  the  pro- 
cess, for  no  general  rule  can  be  given  as  to  the  length  of  time  for 
which  this  stain  must  be  applied ;  it  may  be  ten  seconds,  or  it 
may  be  two  or  three  minutes.     The  only  safe  way  is  to  stain  the 

several  months  after  being  opened,  if  kept  well  corked),  the  method  described 
will  always  succeed  if  well-spread  films  are  used.  This  is  essential.  But  even 
with  bad  films  (and  very  bad  ones  are  sent  me  at  times)  the  results  are  always 
sufificiently  good  to  allow  a  differential  leucocyte  count  to  be  made. 


206  CLINICAL    BACTERIOLOGY    AND   HEMATOLOGY 

film  for  a  quarter  of  a  minute,  wash  it,  and  then  examine  it  under 
the  low  power  of  the  microscope.  If  the  film  is  properly  stained, 
the  nuclei  of  the  leucocytes  will  be  seen  as  blue  points,  which  can 
be  distinguished  with  great  ease  with  the  |-inch  objective.  If 
they  are  not  visible,  the  methylene  blue  must  be  applied  for  about 
a  quarter  of  a  minute  more  and  the  examination  repeated. 
When  the  nuclei  are  seen  to  be  well  stained  the  film  is  dried 
and  mounted. 

This  process  gives  results  which  resemble  those  afforded  by 
Jenner's  stain,  except  that  the  fine  eosinophile  granulations  in  the 
polymorphonuclear  cells  are  always  less  obvious  and  often  quite 
invisible.    It  is  also  suitable  for  malarial  parasites  and  bacteria. 

The  practitioner  is  recommended  to  practise  this  method  of 
staining,  as  it  does  not  require  any  reagents  which  are  not  to  be 
found  in  every  well-stocked  surgery.  The  watery  solution  of 
methylene  blue  which  is  used  as  a  counterstain  for  the  tubercle 
bacillus  and  some  red  ink  are  all  that  are  necessary. 


DIFFERENTIAL  LEUCOCYTE  COUNT 

The  following  varieties  of  leucocytes  are  to  be  recognised,  the 
description  in  each  case  being  taken  from  a  preparation  stained  by 
Jenner's  method. 

A.  Cells  devoid  of  Granules. 

I.  Lymphocytes  (Plate  VII.,  Figs,  i,  2,  3,  4)  are  variable  in  size, 
some  being  about  as  big  as  a  red  corpuscle,  others  nearly  twice  this 
size.  Each  has  a  single  nucleus,  which  is  circular  or  nearly  so, 
and  which  stains  a  deep  blue.  The  protoplasm  forms  a  narrow 
band  round  the  nucleus,  and  also  stains  blue,  often  more  deeply 
than  the  nucleus. 

Variations  of  these  cells  occur.  In  some  cases  the  whole  seems 
to  stain  uniformly,  in  which  case  it  is  probably  a  free  nucleus 
(Plate  VII.,  Fig.  3).  In  others  the  protoplasm  appears  to  be 
studded  with  blue  granules,  which  often  lead  beginners  astray. 
They  are  not  true  granules,  but  knots  in  the  protoplasmic 
network. 

In  healthy  adults  they  average  about  25  per  cent,  of  all  the 
leucocytes  present,  varying  between  22  and  28  per  cent.  In 
childhood  they  are  more  numerous — up  to  60  per  cent. 


DIFFERENTIAL    LEUCOCYTE    COUNT  207 

The  small  forms  are  the  more  numerous,  but  as  no  trustworthy 
diagnostic  information  can  be  drawn  from  the  proportions  of  the 
large  and  small  forms,  they  are  usually  counted  together.  Very 
large  forms  are  often  abundant  in  acute  lymphatic  leucocy- 
thaemia. 

Large  Hyaline  or  Large  Mononuclear  Leucocytes  (Plate  VII., 
Figs.  5,  6). — These  are  the  largest  cells  met  with  in  normal 
blood,  and  may  have  a  diameter  two  and  a  half  that  of  a  red 
corpuscle.  They  have  a  round,  oval,  kidney-shaped,  or  twisted 
nucleus,  which  stains  less  deeply  than  those  of  the  lymphocytes, 
and  has  often  a  purplish  colour.  The  protoplasm  is  relatively 
more  abundant  than  in  the  lymphocytes,  and  stains  very  faintly 
of  a  bluish  or  purplish  colour.  It  often  has  false  granules  similar 
to  those  of  lymphocytes,  but  no  true  ones. 

It  occurs  in  small  numbers  (i  to  4  per  cent.),  and  variations  are 
of  little  diagnostic  importance. 

B.  Cells  containing  True  Granules  in  their  Protoplasm. 

I.  Polynuclear  Leucocytes  (Plate  VII.,  Fig.  7). — These  are  rather 
larger  than  a  red  corpuscle,  and  have  a  nucleus  which  is  twisted 
into  various  irregular  shapes  ;  it  is  often  deeply  lobed,  so  that  it 
appears  to  be  multiple,  but  is  always  really  single  except  in  de- 
generated forms. 

It  contains  in  its  protoplasm  numerous  very  fine  granules  of  a 
substance  which  stains  pink  with  the  eosin  in  Jenner's  stain,  and 
which  are  therefore  considered  by  many  English  pathologists  to 
be  fine  eosinophile  granules.  With  triacid  stain  they  are  a  sort 
of  copper  colour,  quite  unlike  the  large  eosinophile  granules,  and 
are  frequently  spoken  of  as  neutrophile.  The  term  is  a  convenient 
one,  whatever  the  scientific  justification  for  it. 

They  vary  in  numbers  between  rather  wide  limits.  In  the 
adult  70  per  cent,  is  a  fair  average,  but  they  may  be  much  higher, 
and  approach  80  per  cent.  In  childhood  they  are  much  less 
numerous  (30  to  40  per  cent.). 

la.  Myelocytes  or  Neutrophile  Myelocytes. — These  are  the  mother- 
cells  of  the  above,  and  do  not  occur  in  the  blood  in  health,  but 
may  be  found  in  the  bone-marrow.     There  are  two  varieties. 

CorniVs  myelocyte  is  usually  a  very  large  cell  with  a  large,  faint- 
staining  round  or  kidney-shaped  nucleus,  often  placed  decidedly 
to  one  side  of  the  cell  or  even  touching  the  periphery.     The  pro- 


208  CLINICAL    BACTERIOLOGY   AND    HEMATOLOGY 

toplasm  is  relatively  scanty  and  contains  neutrophile  granules, 
often  in  very  small  numbers  and  of  feeble  staining  power.  It  is 
often  necessary  to  use  triacid  stain  to  demonstrate  them  ;  neglect 
to  do  this  may  lead  to  their  being  confounded  with  large  lympho- 
cytes or  hyaline  cells  (Plate  VII.,  Fig.  ii). 

Ehrlich's  myelocyte  is  similar  to  the  above,  but  smaller  ;  it  is 
usually  rather  larger  than  a  polynuclear.  It  has  a  round  or  oval 
nucleus,  which  is  often  central  or  but  slightly  excentric,  and  which 
stains  deeper  than  that  of  Cornil's  myelocyte,  but  not  so  deeply 
as  that  of  a  polynuclear.  Its  neutrophile  granules  are  usually 
distinct  (Plate  VII.,  Fig.  lo). 

Cornil's  myelocyte  is  probably  derived  from  Ehrlich's  by  a 
process  of  degeneration,  especially  dropsy  of  the  nucleus. 

2.  Eosinophile  Leucocytes  (Plate  VII.,  Fig.  8). — These  are  about 
as  large  as  a  polynuclear,  and  have  a  nucleus  which  is  usually 
bilobed,  but  which  may  be  more  distorted.  The  chief  feature  of 
the  cell  is  the  presence  of  numerous  relatively  large  granules 
which  stain  brilliantly  with  the  eosin  :  they  are  spherical  in  shape 
and  very  uniform  in  size.  They  average  about  i  to  4  per  cent,  of 
the  leucocytes  in  health,  but  slightly  higher  or  lower  figures  may 
occur. 

2a.  Eosinophile  myelocytes  (Plate  VII.,  Fig.  12)  do  not  occur  in 
healthy  blood,  and  bear  the  same  relation  to  the  eosinophile  cells 
as  ordinary  myelocytes  to  the  polynuclear  cells.  They  are  dis- 
tinguished from  eosinophiles  by  their  larger  size  and  relatively 
large  circular  nucleus. 

3.  Basophile  Cells  or  Mast  Cells  (Plate  VII.,  Fig.  9). — These  are 
about  as  large  as  polynuclears,  and  have  twisted  (usually  trilohed) 
nuclei,  which  occupy  more  of  the  cell  than  do  those  of  the  poly- 
nuclears. They  have  a  comparatively  small  number  of  granules, 
which  (unlike  those  of  the  other  leucocytes)  vary  in  size  and  stain 
blue  or  purpHsh-blue  with  Jenner.  The  granules  do  not  stain  at 
all  with  triacid. 

They  are  often  not  found  in  persons  in  robust  health,  but  in  the 
average  hospital  patient  occur  in  small  numbers  -  usually  less 
than  f  per  cent. 

-^a.  Large  Mast  Cells  (Plate  VII.,  Fig.  13). — These  occur  only  in 
the  blood  in  myeloid  leucocythaemia,  and  are  very  characteristic 
of  that  condition.  They  may  possibly  be  eosinophile  myelocytes 
with  degenerated  granules. 


DIFFERENTIAL   LEUCOCYTE   COUNT  209 

Method  of  making  a  Differential  Count. 

Having  prepared  a  film  by  any  of  the  methods  previously 
described,  the  next  step  is  to  make  a  differential  count  in  the 
following  way :  Focus  the  film  under  the  microscope,  using  an 
oil-immersion  lens  ;  when  you  have  had  sufficient  experience  it 
is  quite  easy  to  make  the  count  with  a  J-inch  lens,  which  is 
quicker,  but  not  advisable  for  beginners.  Then  note  down  the 
nature  of  each  leucocyte  as  you  come  to  it,  moving  the  film 
across  the  stage  of  the  microscope  from  end  to  end,  and  then 
moving  it  a  little  way  upwards  or  downwards  and  returning  in 
the  opposite  direction,  so  as  never  to  pass  over  the  same  part  of 
the  film  twice.  The  simplest  way  of  noting  down  the  leucocytes 
is  to  assign  single  letters  to  each  variety,  P  for  polynuclear,  E  for 
eosinophile,  etc.,  and  to  put  these  down  in  blocks  of  five  each 
way,  thus : 

P  P  P  LE 

PP  LL  H 

LP  P  P  P 

PL  LPL 

P  P  P  L  P 

In  this  way  you  can  tell  at  any  time  how  many  leucocytes  you 
have  counted.  It  is  much  quicker  to  dictate  the  numbers  to  a 
second  person,  who  takes  them  down  as  described  ;  you  do  not 
then  have  to  look  constantly  from  the  microscope  to  the  paper, 
and  vice  versa^  and  the  whole  process  takes  a  very  few  minutes, 
unless  the  leucocytes  are  very  scanty. 

For  most  clinical  purposes  400  leucocytes  will  be  enough  to 
count,  though  where  very  great  accuracy  is  required  1,000  is 
not  too  many.  Having  counted  the  required  number,  proceed  to 
count  the  numbers  of  P's,  L's,  etc.,  and  reduce  them  to  a 
percentage. 

Whilst  making  the  differential  count  keep  a  sharp  look-out  for 
abnormal  leucocytes,  abnormal  red  corpuscles  (see  p.  213), 
parasites,  etc.  The  collections  of  blood  platelets  which  form 
such  a  prominent  feature  in  some  blood  films  should  not  be 
confounded  with  anything  else,  as  they  are  quite  characteristic, 
though  a  single  blood  platelet  lying  on  the  top  of  a  red  corpuscle 
may  look  very  like  a  young  malaria  parasite.  Each  platelet  is  a 
very  small  mass  which  stains  blue  or  purple  with  Jenner's  stain, 
and  often  appears  hollow  or  irregular  in  shape,  and  they  are  often 
grouped  in  masses  of  quite  large  size. 


210  CLINICAL    BACTERIOLOGY    AND    HiEMATOLOC^ 

Alterations  in  Disease. 

I.  Lymphocytes, — They  may  be  relatively  increased  {e.g.,  to 
figures  above  30  per  cent.)  when  the  total  figures  are  normal  or 
raised.     These  have  different  meanings. 

Lymphocytosis  with  normal  or  lowered  total  counts  occurs  in 
pernicious  anaemia,  typhoid  fever,  uncomplicated  tuberculosis 
(but  not  always),  in  some  cases  of  purpura  (so-called  idiopathic 
purpura),  and  occasionally  in  syphilis,  Hodgkin's  disease,  and 
some  other  diseases. 

Lymphocytosis  with  a  high  total  count  occurs  as  a  normal 
condition  in  infancy,  and  is  accentuated  both  as  regards  the 
percentage  of  lymphocytes  and  the  total  number  of  leucocytes  in 
almost  all  infantile  diseases,  especially  rickets  and  hooping-cough. 

In  adults  a  very  high  total  count  (100,000  or  more)  with  a  very 
high  percentage  of  lymphocytes  (up  to  99*5  per  cent.)  indicates 
lymphatic  leucocythaemia.  Smaller  increases  sometimes  occur 
in  other  diseases. 

A  relative  dimimition  of  the  lymphocytes  frequently  occurs  as  a 
result  of  the  increase  of  other  leucocytes,  rarely  as  a  true  absolute 
diminution. 

Large  Hyaline  Cells.  —  An  increase  or  decrease  of  these  is 
occasionally  observed,  but  so  erratically  that  it  is  of  no  use  in 
diagnosis. 

Polymiclear  Leucocytes. — This  is  the  most  common  cell  to  undergo 
increase,  so  much  so  that  in  the  list  of  causes  of  leucocytosis 
given  on  p.  196  it  may  be  assumed 'that  the  increase  is  due  partly 
or  entirely  to  an  increase  of  polynuclears,  unless  the  opposite  is 
stated  here. 

One  special  case  needs  a  reference.  In  severe  sepsis  with  a 
normal  or  diminished  polynuclear  count  it  is  usual  for  the 
polynuclears  to  be  relatively  increased,  just  as  they  are  when 
the  total  count  is  raised.  This  is  very  important  in  diagnosis, 
for  in  cases  where  sepsis  is  suspected  a  normal  leucocyte  count 
must  always  be  followed  by  a  differential  count,  and  if  there  is  a 
relative  increase  {e.g.^  85  per  cent,  or  more)  of  polynuclears  it 
must  be  regarded  as  a  bad  sign.  A  relative  increase  of  poly- 
nuclears with  a  normal  or  moderately  raised  total  count  may  also 
occur  in  malignant  disease. 

A  form  of  degeneration  of  the  polynuclears  requires  notice,  as 
it  is  of  some  practical  value.     This  is  the  iodine  or  glycogenic 


DIFFERENTIAL   LEUCOCYTE   COUNT  211 

reaction.  To  test  for  it  make  a  film  in  the  ordinary  way,  dry 
and  mount  it  without  fixation  in  the  following  mixture : 

Iodine i  part. 

Iodide  of  potassium       -         -         -         -         3  parts.       ^ — 
Saturated  watery  solution  of  gum  acacia    loo  parts. 

This  should  not  be  used  more  than  a  fortnight  after  it  has  been 
made.  A  simpler  but  equally  good  method  is  to  expose  the  film 
to  the  fumes  of  solid  iodine  for  a  few  hours.  This  can  be  done  by 
fixing  the  film  (without  previous  treatment  of  any  sort)  at  the  top 
of  a  wide-mouthed  bottle  containing  the  substance ;  it  is  then 
mounted  in  oil  or  balsam.  Allow  it  to  act  for  a  quarter  of  an 
hour  or  more,  then  examine  with  a  yV-inch  lens  in  a  white  light 
— daylight  if  possible.  In  cases  where  the  reaction  is  present 
a  variable  number  of  the  polynuclears  will  be  found  to  contain 
granules  or  masses  of  a  reddish-brown  or  mahogany  colour  ; 
sometimes  there  are  large  masses,  and  sometimes  almost  the 
whole  of  the  protoplasm  appears  brown. 

The  importance  of  this  reaction  is  that  it  usually  occurs,  and 
is  indeed  very  marked,  in  the  cases  of  severe  sepsis  in  which 
there  is  no  increase  of  the  total  leucocyte  count  (see  p.  198),  as 
well  as  in  suppuration.  It  occurs  in  other  conditions,  such  as 
pneumonia,  hooping-cough,  uraemia,  etc.  In  many  cases  a  small 
percentage  only  of  the  cells  is  aff'ected,  and  a  good  search  must 
be  made. 

The  fact  that  it  occurs  in  so  many  diseases  detracts  somewhat 
from  its  value  in  diagnosis,  but  when  the  question  is  simply  the 
presence  or  absence  of  pus — e.g.j  in  appendicitis — a  positive  result 
will  usually  indicate  that  suppuration  has  occurred,  and  vice  versa. 
Hence  it  is  sometimes  of  value  when  the  leucocyte  count  yields 
uncertain  results — i.e.,  figures  between  16,000  to  20,000  per  cubic 
millimetre. 

Myelocytes. — Ehrlich's  myelocytes  occur  in  small  numbers  in 
many  infectious  diseases,  especially  diphtheria,  and  occasionally 
in  forms  of  anaemia,  but  they  are  only  present  in  large  numbers 
in  myeloid  leucocythaemia.  Cornil's  myelocytes  are  practically 
limited  to  the  latter  condition,  where  the  two  forms  frequently 
together  make  up  60  per  cent,  of  all  leucocytes. 

Eosimphiles. — An  increase  of  eosinophiles  (eosinophilia)  occurs 
to  a  small  extent  in  numerous  diseases,  and  is  of  diagnostic 
importance  in  the  following  : 

I.  In  diseases  due  to  animal  parasites.     Here  the  increase  may 

14 — 2 


212  CLINICAL    BACTERIOLOGY   AND   HEMATOLOGY 

be  very  great,  as  in  trichinosis,  where  the  eosinophiles  usually 
form  40  to  80  per  cent,  of  all  leucocytes,  a  fact  of  great  import- 
ance, and  absolutely  distinguishing  the  disease  from  typhoid 
fever,  rheumatism,  and  other  diseases  which  may  be  confounded 
with  it ;  rarely,  however,  there  may  be  no  eosinophilia,  so  that 
its  absence  does  not  definitely  exclude  the  disease.  In  bilharzia 
disease  there  is  often,  though  not  always,  a  mild  eosinophilia. 
There  is  practically  always  an  increase  in  ankylostomiasis.  It 
may  be  moderate,  or  may  reach  70  per  cent.  This  fact  enables 
infected  individuals  to  be  picked  out  of  a  gang  of  workmen  with 
much  less  trouble  than  by  an  examination  of  the  faeces.  In 
diseases  due  to  other  intestinal  worms  (ascarides,  oxyuris,  taeniae), 
there  may  or  may  not  be  eosinophilia. 

In  hydatid  disease  there  is  frequently  a  moderate  eosinophilia, 
and  sometimes  a  great  one.  This  is  important  in  the  diagnosis 
between  hydatid  and  abscess  of  the  liver,  since  in  the  latter 
condition  the  eosinophiles  are  usually  scanty.  In  such  a  case 
the  higher  the  count  is  above  4  per  cent.,  the  more  likely  is  the 
disease  to  be  hydatid,  and  vice  versa. 

In  the  only  case  of  cysticercus  I  have  seen  the  eosinophiles 
ranged  between  5  and  7  per  cent. 

2.  In  extensive  skin  diseases,  especially  pemphigus  and 
urticaria,  there  is  often  a  great  increase,  but  it  occurs  in  so  many 
conditions  that  its  diagnostic  value  is  but  slight  (see  p.  232). 

3.  Diseases  of  the  lungs. — In  true  asthma  there  is  during  the 
paroxysms,  and  for  a  short  time  after  them,  a  very  decided 
increase — to  10  per  cent,  or  so.  The  cells  in  the  sputum,  too, 
are  practically  all  eosinophiles.  This  does  not  occur  in  the  other 
spasmodic  diseases  which  so  mimic  true  asthma,  and  is  often  of 
critical  importance  in  the  diagnosis.  Eosinophiles  are  never 
found  in  the  sputum  in  tuberculosis. 

4.  Myelogenous  leucocythcemia. — Here  there  is  an  enormous  abso- 
lute increase  in  the  total  numbers  of  the  eosinophiles,  counting  the 
eosinophile  myelocytes  with  them  ;  the  percentage  may  not  be 
greater  than  in  health. 

This  is  one  of  the  most  constant  signs  of  the  condition. 

A  decrease  of  eosinophiles  occurs  in  nearly  all  inflammatory 
leucocytoses,  especially  in  pneumonia,  where  a  careful  search  over 
many  films  may  fail  to  reveal  the  presence  of  a  single  cell.  Their 
reappearance  in  any  of  these  diseases  is  of  distinctly  good  omen, 
and  one  on  which  I  place  a  good  deal  of  reliance.     It  is  not  of 


DIFFERENTIAL   LEUCOCYTE    COUNT  213 

much  value  in  pneumonia,  as  it  does  not  occur  till  after  the  crisis, 
but  in  chronic  suppurative  diseases,  etc.,  a  gradual  increase  in 
the  eosinophiles  often  heralds  improvement.  Neusser  holds  that 
the  same  is  true  in  tuberculosis  also.  - 

Eosinophile  Myelocytes.— The  presence  of  these  is  practically 
diagnostic  of  myelogenous  leucocythaemia. 

Mast  Cells. — These  only  occur  in  fractional  percentages  in  any 
disease,  except  myeloid  leucocythaemia,  where  5  or  lo  per  cent,  is 
common  ;  this  is  perhaps  the  most  definite,  constant,  and  readily 
recognised  sign  of  the  disease.  The  large  forms,  with  circular  or 
kidney-shaped  nuclei,  are  practically  only  seen  in  this  condition. 

Morphological  Changes  in  the  Red  Corpuscles. 

Normal  red  corpuscles  (Plate  VIII.,  Fig.  i)  hardly  need 
description,  and  the  practitioner  can  readily  make  himself 
familiar  with  their  appearance.  They  are  strongly  oxyphile, 
staining  pink  with  the  eosin  in  Jenner's  stain,  and  with  acid 
fuchsin  in  the  triacid.  It  is  especially  important  to  become 
familiar  with  their  size,  since  modifications  in  this  respect  are 
very  important  in  the  diagnosis  of  certain  forms  of  anaemia. 

Abnormal  Forms  of  Red  Corpuscles. 

Two  great  classes  are  to  be  recognised — those  which  retain 
the  characters  of  a  normal  red  corpuscle,  in  that  they  have  no 
nuclei,  and  those  which  resemble  the  embryonic  corpuscles  in 
being  nucleated.  The  form^er,  where  they  have  special  designa- 
tions, have  names  terminating  in  -cyte  (microcyte,  megalocyte, 
etc.,  based  on  the  type  of  the  normal  corpuscle,  which  is  called 
erythrocyte,  xanthocyte,  or  normocyte,  convenient  terms  that 
have  never  gained  wide  currency),  whilst  the  nucleated  forms 
have  names  ending  in  -blast. 

Abnormal  Non-nucleated  Forms  (-cytes). 

1.  Microcytes  (Plate  VIII.,  Fig.  4)  are  red  corpuscles  which  are 
decidedly  below  the  normal  in  size,  but  are  otherwise  normal. 
They  are  indications  of  anaemia,  but,  as  they  may  occur  in  any 
severe  anaemia,  and  do  not  serve  to  indicate  its  type,  their  recog- 
nition is  not  a  matter  of  much  importance. 

2.  Megalocytes  ov  Macvocytes  (Plate  VIII.,  Fig.  5). — These  are 
large  corpuscles,  and   the   name   should   be   restricted  to  forms 


214  CLINICAL    BACTERIOLOGY   AND    H.EMATOLOGY 

that  are  at  least  one  and  a  half  times  as  broad  as  a  normal 
corpuscle  ;  they  may  be  much  larger. 

It  is  important  to  learn  to  recognise  them,  since  their  presence 
is  an  almost  constant  sign  of  pernicious  anaemia,  and  it  is  rare 
to  find  them  in  any  considerable  numbers  in  other  conditions. 
When  you  are  frequently  examining  blood-films  with  the  same 
lens  and  eyepiece,  you  will  soon  be  able  to  pick  out  forms  of 
abnormal  size.  When  you  have  not  had  this  experience  it  is 
advisable  to  make  a  film  of  normal  blood  for  comparison,  and  to 
look  first  at  one  and  then  at  the  other.  A  still  better  method 
(due  to  Shattock)  is  to  mount  the  two  films  (the  normal  and 
abnormal,  both  stained)  face  to  face,  and  then  to  mount  the  pair 
on  a  slide,  and  examine  them  with  an  oil-immersion  lens  ;  a  slight 
turn  of  the  fine  adjustment  will  enable  you  to  pass  at  once  from  the 
one  to  the  other,  and  to  compare  the  diameters  of  the  corpuscles 
with  some  degree  of  accuracy.  Another  useful  method  is  to 
employ  the  counting-chamber  of  the  haemocytometer,  and  an 
idea  of  the  diameter  of  the  reds  should  be  gained  in  all  cases  of 
pernicious  anaemia  whilst  the  count  is  being  made.  The  diameter 
of  one  of  the  small  squares  is  almost  exactly  six  times  that  of  a 
normal  red  corpuscle.  If  you  look  at  the  squares  with  a  central 
ruling,  you  will  be  able  to  compare  any  corpuscles  which  may  be 
in  them  with  the  semi-diameter ;  the  one  should  be  one -third  of 
the  other,  and  you  may  consider  as  a  megalocyte  any  corpuscle 
which  is  one-half  or  more  of  this  semi-diameter. 

3.  Poikilocytes  (Plate  VIII.,  Fig.  6)  are  deformed  corpuscles, 
and  are  typically  shaped  like  a  pear,  but  may  be  kidney-shaped 
or  quite  irregular.  They  may  be  about  as  large  as  a  normal 
corpuscle,  or  smaller,  or  larger,  and  they  may  stain  abnormally. 
Poikilocytes  are  more  common  in  pernicious  anaemia  than  in 
other  diseases,  but  are  not  of  much  diagnostic  importance,  since 
they  only  occur  in  advanced  stages  of  the  disease,  long  after  the 
diagnosis  should  have  been  made.  To  recognise  them,  put  a 
small  drop  of  blood  on  a  perfectly  clean  slide,  apply  a  cover-glass, 
and  examine  at  once  ;  do  not  identify  poikilocytes  in  dried  films 
or  in  the  counting-chamber  of  the  haemocytometer  until  you  have 
had  a  good  deal  of  experience,  as  accidentally  injured  and  con- 
torted forms  may  occur  in  either. 

4.  Polychvomatophil  Degeneration  (Plate  VIII.,  Fig.  3). — In  this 
condition  the  corpuscle  (which  may  otherwise  be  normal  or 
abnormal  in  other  ways),  instead  of  being  strictly  acidophile  in 


DIFFERENTIAL    LEUCOCYTE    COUNT  215 

its  staining  reactions,  stains  with  the  basic  stain  to  a  greater  or 
less  extent ;  thus,  with  Jenner's  stain  it  stains  a  variable  mixture 
of  pink  (from  the  eosin)  and  blue  (from  the  methylene  blue).  It 
may  be  lilac,  purplish,  or  almost  pure  blue.  The  change  is 
readily  recognisable  in  ordinary  films  stained  by  Jenner's  method. 

It  is  especially  common  in  pernicious  anaemia  and  in  von 
Jaksch's  anaemia  of  children,  but  may  occur  in  almost  any  form 
of  anaemia,  if  very  severe.  It  is  not  a  very  important  diagnostic 
feature,  but  its  presence  always  constitutes  a  bad  sign. 

5.  Granular  degeneration  (Plate  VIII.,  Fig.  2)  takes  the  form  of 
numerous  granules  of  varying  size,  which  occur  in  the  red  cor- 
puscles, and  which  stain  almost  black  with  the  basic  portion  of 
the  stain  ;  the  rest  of  the  corpuscle  often  shows  polychromatophil 
degeneration. 

It  occurs  also  in  any  severe  anaemia,  especially  in  von  Jaksch's 
anaemia,  where  corpuscles  in  which  it  occurs  may  be  extremely 
plentiful.  Except  where  this  occurs  it  is  not  of  much  diagnostic 
importance,  unless  it  is  true  that  it  occurs  as  a  very  early  and 
constant  sign  in  lead-poisoning. 

Nucleated  Forms  (-blasts). 

I.  Normoblasts  (Plate  VIII.,  Figs.  7, 8,  9). — These  are  corpuscles 
which  resemble  the  normal  in  shape  and  size,  but  which  have  a 
nucleus.  This  is  central,  large  in  proportion  to  the  corpuscles, 
surrounded  by  a  comparatively  narrow  band  of  stroma,  and 
circular;  in  some  cases  it  is  double  or  multiple.  Normoblasts  can 
usually  be  recognised  with  ease  from  any  other  cells  which  occur  in 
the  blood,  from  the  fact  that  the  nucleus  stains  very  deeply — more 
deeply  than  any  other  found  in  the  blood.  It  frequently  happens 
that  the  narrow  ring  of  stroma  may  show  polychromatophil  de- 
generation and  stain  blue  ;  in  this  case  it  is  difficult  to  distinguish 
the  cell  from  a  lymphocyte,  but  the  deeply  stained,  almost  black, 
nucleus  should  prevent  mistakes. 

Normoblasts  are  the  cells  from  which  the  normal  blood- 
corpuscles  are  formed,  but  in  health  they  are  confined  to  the 
bone-marrow,  except  in  very  young  infants,  in  whom  a  very  few 
may  be  found  in  the  circulation.  Their  presence  in  the  blood  of 
older  persons  indicates  an  anaemia  of  some  severity,  and  that  thia 
anaemia  is  being  combated  in  a  normal  way ;  the  bone-marrow  is 
so  active  that  some  of  its  normoblasts  overflow  into  the  circulating 
blood.     They  are  therefore  rather  a  good  sign   than  otherwise. 


2l6  CLINICAL    BACTERIOLOGY   AND    HiEMATOLOGY 

Occasionally  you  may  find  them  in  very  large  numbers  in  the 
blood  of  a  case  of  anaemia,  especially  in  chlorosis :  this  is  called  a 
"blood-crisis,"  and  when  it  occurs  the  patient  will  improve  very 
rapidly.  If  you  want  to  count  their  numbers,  the  simplest  way  is 
as  follows  :  Count  the  leucocytes  in  the  way  already  described, 
and  calculate  the  number  per  cubic  millimetre.  Then  take 
a  stained  film  and  count  400  or  500  leucocytes,  noting  how  many 
normoblasts  you  see  whilst  doing  so.  A  simple  calculation  will 
give  the  number  of  normoblasts  per  cubic  millimetre.  Thus, 
if  there  are  8,000  leucocytes  per  cubic  millimetre,  and  72  normo- 
blasts are  seen  whilst  counting  500  leucocytes,  the  total  number 

of  normoblasts  per  cubic  millimetre  is  7^  ^    >QQQ  ^  1,152. 

500 

2.  Megalohlasts  (Plate  VIII.,  Figs.  10,  11,  12). — The  recog- 
nition of  these  is  of  the  utmost  importance,  as  if  they  are 
present  in  any  appreciable  numbers  in  the  blood  of  an  adult 
the  diagnosis  of  pernicious  anaemia  is  almost  a  certainty,  and 
if  a  single  example  is  found  the  diagnosis  is  highly  probable. 
They  occur  very  rarely  in  adults  in  other  conditions,  but  are 
more  common  in  severe  anaemia  in  childhood,  especially  in 
von  Jaksch's  anaemia,  where  they  may  be  plentiful. 

In  size  megaloblasts  resemble  megalocytes,  but  may  be  even 
'arger ;  very  large  forms  are  called  gigantoblasts,  and  may  have 
a  diameter  nearly  three  times  as  great  as  a  normal  corpuscle. 
A  megaloblast  has  a  nucleus  which  is  usually  larger  than  that  of 
a  normoblast,  though  it  may  be  smaller  in  proportion  to  the  size  of 
the  cell ;  it  is  sometimes  double  or  multiple.  It  stains  much  less 
deeply  than  the  nucleus  of  a  normoblast — in  fact,  it  may  stain  so 
faintly  that  it  is  overlooked.  Except  for  this  the  only  mistake 
commonly  made  by  beginners  is  to  confuse  a  megaloblast  with 
polychromatophilic  stroma  (which  frequently  occurs)  with  a  large 
hyaline  leucocyte. 

DIAGNOSTIC  APPLICATIONS  OF  THE  BLOOD- 
COUNT  AS  A  WHOLE 

In  this  chapter  a  brief  outline  of  the  chief  practical  applications 
of  the  blood-count  will  be  given,  with  especial  reference  to  cases 
where  it  is  of  use  in  the  discrimination  between  two  diseases 
which  are  difficult  to  distinguish  clinically. 

HAEMORRHAGE. — After  a  severe  haemorrhage  there  is  at  first  no 
alteration  of  the  blood ;  the  total  volume  is  diminished,  but  the 


DIAGNOSTIC   APPLICATIONS    OF    THE    BLOOD-COUNT     217 

part  that  remains  is  normal.  In  a  very  short  time,  however,  fluid 
is  absorbed  from  the  tissues  so  as  to  make  up  the  normal  volume, 
and  in  consequence  the  red  corpuscles  and  haemoglobin  fall  in  the 
same  proportion.  With  this  there  is  usually  a  leucocytosis,  high 
figures  (20,000  or  more)  being  sometimes  reached  ;  do  not  forget 
this  in  dealing  with  a  blood-count  in  a  patient  who  has  recently 
had  a  haemorrhage.  As  the  process  of  regeneration  continues,  the 
improvement  shows  itself  first  in  an  increase  of  the  red  corpuscles, 
so  that  the  colour-index  falls  slightly  (to  about  0-9). 

The  anaemia  from  repeated  haemorrhages  is  one  of  the  varieties 
of  secondary  anaemia. 

Secondary  Anemia. — This  term  is  used  for  anaemia  which  is 
due  to  any  definitely  recognised  cause — haemorrhage,  malnutrition, 
sepsis,  intoxications  {e.g.,  lead-poisoning),  etc. — that  is  to  say,  it 
includes  all  cases  of  anaemia  except  those  of  unknown  pathology, 
such  as  chlorosis  and  pernicious  anaemia. 

In  secondary  anaemia  there  is  a  reduction  of  red  corpuscles  to 
an  extent  dependent  on  the  potency  and  continuance  of  the  cause, 
and  a  slightly  greater  reduction  of  the  haemoglobin,  so  that  the 
colour-index  is  lowered.  It  does  not  usually  fall  below  0*7, 
and  0-8  may  be  taken  as  a  fair  average.  Anything  below  this  is 
rarely  met  with  apart  from  chlorosis.  The  red  corpuscles  are 
usually  practically  normal  in  appearance  ;  normoblasts  are  rare, 
and  their  presence  is  a  good  sign. 

The  leucocytes,  especially  the  polynuclears,  are  usually  slightly 
increased,  and  this  is  the  chief  or  only  means  of  distinguishing  a 
secondary  anaemia  with  low  colour-index  from  chlorosis,  in  which 
the  leucocytes  are  normal  or  reduced. 

Pernicious  Anemia. — The  feature  which  usually  first  raises 
suspicion  of  pernicious  anaemia  is  the  high  colour -index.  It  is 
usually  over  i,  and,  taking  the  average  of  a  number  of  cases, 
it  increases  in  proportion  to  the  amount  of  reduction  of  the 
corpuscles,  thus : 


Red  Corpuscles. 

Average  Colour-index. 

Pernicious.      |      Secondary. 

1 

Under  500.000 
500,000  to  1,000,000 
1,000,000  to  2,000,000 
2,000,000  to  3,000,00D 
3,000,000  to  4,000,000 

x-6 

1-4 

1-23 
I '2 
0-99 

077 
0-82 
0-82 

2l8  CLINICAL    BACTERIOLOGY   AND    HiEMATOLOGY 

When  you  find  a  case  with  a  high  colour-index,  it  should 
immediately  raise  a  suspicion  of  pernicious  anaemia.  Look 
again  at  the  preparation  in  which  you  have  counted  the  reds  in 
the  Thoma-Zeiss  haemocytometer,  and  look  for  unusually  large 
corpuscles  (megalocytes),  which,  according  to  Ewing,  should  form 
35  per  cent,  of  all  corpuscles,  or  the  diagnosis  is  to  be  made  with 
caution.  Then  count  the  leucocytes  in  the  same  preparation. 
Leucopenia  is  very  characteristic ;  if  the  number  exceeds  6,000, 
pernicious  anaemia  is  unlikely,  unless  inflammatory  complications 
are  present.  Then  make  a  differential  count  on  a  stained  film, 
looking  out  for  megaloblasts  and  nonnohlasts  as  you  do  so ;  in 
pernicious  anaemia  there  is  almost  always  a  relative  lymphocytosis, 
and  the  diagnosis  is  unlikely  with  the  lymphocytes  much  below 
40  per  cent.  If  you  have  not  yet  seen  a  megaloblast,  continue  to 
search  for  them,  as  they  are  usually  present  in  pernicious  anaemia 
of  moderate  severity,  and  comparatively  rare  in  other  conditions, 
except  in  children.  The  significance  of  the  discovery  of  a  single 
megaloblast  will  depend  on  the  other  findings  ;  if  these  point  to 
pernicious  anaemia,  the  megaloblast  may  be  taken  as  clinching 
the  diagnosis,  but  if  they  are  not  of  this  nature  its  importance  is 
much  less.  Do  not  exclude  pernicious  anaemia  because  no 
megaloblasts  are  found.  According  to  Ehrlich  and  others  they 
always  exceed  the  normoblasts  in  numbers,  but  this  is  not  a  safe 
guide,  as  in  some  cases  you  may  find  normoblasts  alone  on  some 
occasions,  and  megaloblasts  and  normoblasts  in  differing  pro- 
portions on  others.  Polychromatophil  and  granular  degeneration 
and  poikilocytosis  are  common  in  advanced  stages  of  the  disease, 
but  the  diagnosis  ought  to  have  been  made  before  their  appearance. 

The  diagnosis  from  secondary  ancemia  rests  on  the  high  colour- 
index,  the  leucopenia,  lymphocytosis,  nucleated  corpuscles  and 
megalocytes,  and  is  usually  easy.  Anaemia  associated  with 
intestinal  parasites  may  resemble  idiopathic  pernicious  anaemia  in 
every  respect  except  in  that  the  former  is  accompanied  by 
eosinophilia.  In  pernicious  anaemia  the  eosinophiles  are  usually 
low,  and  if  they  exceed  4  per  cent,  the  faeces  should  be  searched 
for  the  ova  of  parasites  (especially  ankylostoma,  bothriocephalus 
and  oxyuris).  The  anaemia  of  carcinoma  of  the  stomach  and  other 
gastric  diseases  may  closely  resemble  pernicious  anaemia,  but  in 
most  cases  there  is  a  high  leucocyte  count,  with  increase  of 
polynuclears  and  diminution  of  lymphocytes. 

Chlorosis. — Here  there  is  a  moderate  reduction  of  the  red 


I 


DMGNOSTIC   APPLICATIONS    OF   THE    BLOOD-COUNT        2ig 

corpuscles  and  a  great  reduction  of  the  haemoglobin  ;  the  colour- 
index  falls,  therefore,  and  0-5  may  be  taken  as  an  average, 
though  much  lower  figures  occur.  The  corpuscles  are  pale,  but 
abnormal  forms  are  rare.  Normoblasts  are  very  rare,  but  when_ 
they  occur  usually  herald  a  rapid  improvement.  The  leucocytes 
are  normal  in  numbers,  or  there  may  be  leucopenia. 

It  may  be  confounded  with  various  forms  of  secondary  anaemia, 
but  in  them  the  colour-index  is  usually  higher,  and  the  leucocytes, 
especially  the  polynuclears,  tend  to  be  increased. 

Myelogenous  Leucocyth^mia  ("  spleno-medullary"). — There 
is  an  enormous  increase  in  leucocytes,  which  in  an  average  case 
may  amount  to  400,000.  All  varieties  of  leucocytes  are  increased 
in  absolute  numbers,  but  the  increase  mainly  affects  the  poly- 
nuclears and  the  eosinophiles  ;  the  lymphocytes  are  relatively  so 
scanty  that  they  may  be  difficult  to  find.  In  addition,  there  are 
abnormal  cells  :  eosinophile  myelocytes  and  large  cells  with 
basophile  granulations,  cells  occurring  in  practically  no  other 
disease,  and  both  Ehrlich's  and  Cornil's  myelocytes. 

There  is  usually  anaemia  of  the  secondary  type,  and  normoblasts 
are  numerous. 

The  whole  picture  is  most  characteristic,  and  can  hardly  be 
mistaken  for  anything  else. 

Lymphatic  Leucocyth^mia. — There  is  no  difficulty  in  the 
recognition  of  a  typical  case  of  chronic  lymphatic  leucocy- 
thaemia  ;  the  leucocytes  are  enormously  increased  (100,000  to 
1,000,000),  and  consist  almost  entirely  of  lymphocytes  (often  up 
to  99  per  cent.)  :  in  many  cases  the  large  forms  predominate. 
There  is  also  a  varying  degree  of  secondary  anaemia. 

In  an  adult  these  appearances  are  quite  characteristic,  but  in 
childhood  similar  counts  may  be  seen  in  a  variety  of  conditions, 
such  as  hooping-cough,  broncho-pneumonia,  etc.,  though  it  is 
rare  to  find  a  figure  as  high  as  100,000. 

In  some  acute  cases  of  lymphatic  leucocythaemia  similar 
appearances  occur.  In  others  the  increased  percentage  of 
lymphocytes  is  present,  but  not  the  total  increase  ;  thus,  in  one 
case  fatal  in  a  few  weeks  I  never  found  more  than  10,000 
leucocytes,  in  which  the  lymphocytes  varied  between  76  per  cent, 
and  100  per  cent.  These  cases  are  very  difficult  to  diagnose 
from  acute  tuberculosis  of  the  lymphatic  glands.  In  the  latter 
case  the  total  count  may  be  expected  to  be  low,  in  the  former 
slightly  raised ;  in  tubercle  the  percentage  of  lymphocytes  rarely 


220  CLINICAL    BACTERIOLOGY   AND    HEMATOLOGY 

exceeds  50,  whilst  75  per  cent,  at  least  would  be  required  before 
the  condition  under  discussion  could  be  diagnosed. 

Other  diseases  (especially  acute  purpura  haemorrhagica)  give 
very  high  lymphocytoses  in  adults,  but  as  they  are  quite  different 
clinically  the  blood-count  would  not  be  misleading. 

Hodgkin's  Disease. — There  is  little  doubt  that  several  diseases 
are  included  under  this  heading.  In  the  true  Hodgkin's  disease 
there  is  at  first  no  change  in  the  blood,  not  even  anaemia  ;  in  one 
case  I  examined  at  intervals  for  over  a  year  the  red  corpuscles 
were  always  above  5,000,000,  and  the  haemoglobin  above  100  per 
cent.  Later,  there  is  anaemia  of  secondary  type,  with  a  slight 
leucocytosis,  with  or  without  a  moderate  increase  of  polynuclears. 
In  other  cases  there  may  be  anaemia  from  the  first,  a  normal  or 
diminished  number  of  leucocytes,  and  a  relative  increase  of 
lymphocytes  of  moderate  amount ;  these  cases  are  probably  more 
closely  allied  to  lymphatic  leucocythaemia,  and  for  them  the  term 
"  pseudo-leukaemia  "  might  be  used.  I  believe  them  to  be  of  more 
rapid  course  than  true  Hodgkin's,  but  the  two  cannot  be  definitely 
separated  on  clinical  grounds  alone. 

The  diagnosis  between  these  forms  of  enlarged  glands  and 
those  due  to  tuberculosis  cannot  be  made  by  a  blood-count.  The 
opsonic  index  to  tubercle  may  be  of  assistance. 

Lymphosarcoma  is  also  associated  with  a  practically  normal 
blood  condition,  and  cannot  be  diagnosed  from  aleukaemic  leuco- 
cythaemia and  Hodgkin's  disease. 

Splenic  Anemia. — It  is  very  doubtful  whether  this  disease  is 
really  a  distinct  entity,  and  it  is  certain  that  many  of  the  reported 
cases  have  nothing  in  common  but  the  accidental  and  not  unusual 
concomitance  of  anaemia  and  an  enlarged  spleen.  In  the  cases  in 
which  the  name  may  fairly  be  applied  there  is  idiopathic  enlarge- 
ment of  the  spleen,  with  anaemia ;  the  latter  is  usually  of  medium 
grade,  figures  under  2,500,000  being  uncommon,  and  the  colour- 
index  is  moderately  low  (07  to  o'g).  In  severe  cases  there  may 
be  numerous  normoblasts,  poikilocytes,  and  polychromatophil 
degeneration  of  the  red  corpuscles.  The  only  feature  that  can  be 
considered  as  characteristic  is  the  frequent  presence  of  leiicopenia 
with  relative  lymphocytosis  ;  there  may  be  a  few  myelocytes. 

It  may  be  confounded  clinically  with  myelogenous  leucocy- 
thaemia or  pernicious  anaemia,  but  is  readily  distinguished  by  the 
blood-count.  In  secondary  anaemia  with  an  enlarged  spleen  (such 
as  occurs  in  malignant  disease,  infective  processes,  etc.)  there  will 


DIAGNOSTIC    APPLICATIONS   OF   THE    BLOOD-COUNT     221 

probably  be  a  polynuclear  leucocytosis.  Hodgkin's  disease  with 
an  enlarged  spleen  and  without  palpable  glands  may  be  indis- 
tinguishable from  splenic  anaemia,  and  probably  some  of  the 
recorded  cases  have  been  of  this  nature.  — 

Anemia  in  Infancy. — The  rules  for  the  interpretation  of  blood- 
counts  in  adults  are  not  applicable  in  infancy,  where  the  conditions 
of  blood  formation  are  so  different.  In  particular  the  presence  of 
nucleated  red  corpuscles,  and  especially  of  megaloblasts,  is  of  little 
importance,  and  frequently  occurs  in  conditions  insufficient  to 
call  for  their  appearance  in  older  patients.  The  colour- index  is 
extremely  variable  ;  it  usually  tends  to  be  very  low,  although  this 
does  not  indicate  a  disease  having  any  connection  with  chlorosis, 
a  disease  which  does  not  occur  in  infancy.  On  the  other  hand,  a 
high  colour-index  is  not  infrequent,  and  does  not  necessarily 
point  to  pernicious  anaemia,  which  is  excessively  rare.  Lastly, 
degenerative  changes,  such  as  poikilocytosis,  granular  degeneration, 
and  polychromatophilia,  are  very  common,  occur  in  comparatively 
mild  grades  of  anaemia,  and  have  not  the  serious  import  they  have 
in  the  adult. 

Leucocytosis  in  Anamia  in  Infants. — This  is  very  common,  20,000 
to  60,000  being  frequently  met  with  ;  in  most  cases  there  is  a 
predominance  of  lymphocytes,  and  the  presence  of  myelocytes  is 
quite  common,  rendering  the  diagnosis  from  leucocythaemia  a 
matter  of  some  difficulty.  As  a  general  rule,  the  presence  of  a 
high  leucocytosis  in  infantile  anaemia  is  a  bad  sign,  and  indicates 
a  worse  prognosis  than  if  it  is  absent. 

Secondary  Ancsmia  in  Infancy  {Syphilis,  Rickets ,  Scurvy,  Tubercle, 
etc.), — There  are  no  characteristic  changes  by  which  the  different 
causes  of  secondary  anaemia  can  be  recognised.  The  colour-index 
is  usually  low  (especially,  perhaps,  in  syphilis),  normoblasts  are 
not  uncommon,  an  occasional  megaloblast  may  be  seen,  and  there 
is  often  leucocytosis,  with  increase  of  lymphocytes. 

Von  Jaksch's  Anmnia  {Anmnia  Infantum,  Pseudo-leukcsmia). — It  is 
uncertain  whether  this  is  to  be  considered  as  a  definite  disease,  as 
an  intermediate  form  between  pernicious  anaemia  and  leucocy- 
thaemia, or  as  a  form  of  secondary  anaemia  with  somewhat 
characteristic  blood  changes.  I  am  rather  inclined  to  the  belief 
that  it  does  represent  a  definite  blood  disease,  but  that  it  is 
frequently  associated  with,  and  perhaps  due  to,  other  diseases 
such  as  syphilis,  rickets,  tubercle,  or  gastro-intestinal  diseases. 
The  blood  changes  are — (i)  An  excessive  grade  of  anaemia,  usually 


222  CI.INICAL    BACTERIOLOGY    AND    HEMATOLOGY 

associated  with  a  low  colour-index  ;  in  some  cases,  however,  it 
may  be  high,  and  I  have  seen  it  as  high  as  i"8.  (2)  High 
leucocytosis,  often  50,000  or  more,  with  extraordinary  changes  in 
the  leucocytes,  so  that  they  can  hardly  be  classified  on  the  usual 
lines  ;  myelocytes  are  not  uncommon.  (3)  Striking  and  profound 
changes  in  the  red  corpuscles,  the  most  important  being  the 
presence  of  numerous  normoblasts,  many  of  which  show  dividing 
miclei  (Plate  VI 1 1.,  Fig.  8)  ;  megaloblasts  and  atypical  forms 
occur,  but  are  less  numerous.  The  non-nucleated  red  corpuscles 
show  all  forms  of  degeneration,  poikilocytes,  megalocytes,  and 
microcytes  being  present,  whilst  many  are  affected  with  granular 
basophilia  (Plate  VIII.,  Fig.  2)  or  polychromatophilia  (Plate  VIII., 
Fig.  3).  In  severe  cases  the  majority  of  the  red  corpuscles  may 
be  abnormal,  and  the  appearance  of  the  stained  films  is  very 
extraordinary. 

The  prognosis  of  these  cases  is  fairly  good  if  proper  treatment 
be  adopted — much  better  than  in  the  primary  blood  diseases  with 
which  they  might  be  confounded. 

Sepsis,  Suppuration,  and  SEPTic^MtA. — In  most  cases  of  infec- 
tion with  septic  bacteria,  whether  local  or  general,  there  is  marked 
leucocytosis,  due  especially  to  an  increase  in  the  polynuclear 
leucocytes.  For  instance,  in  an  ordinary  case  of  appendicitis 
of  average  severity  we  may  expect  the  number  of  leucocytes  to 
rise  gradually  to  25,000  or  30,000,  about  90  per  cent,  being  poly- 
nuclears.  At  the  same  time  there  is  usually  a  moderate  fall  in 
the  amount  of  haemoglobin  and  in  the  number  of  red  corpuscles. 
In  practice  we  have  to  consider  three  types,  in  each  of  which  the 
blood-counts  vary. 

I,  Very  Severe  Cases.- — Puerperal  and  other  forms  of  septicaemia, 
general  septic  peritonitis  due  to  very  virulent  bacteria,  especially 
in  an  enfeebled  patient,  etc. 

Here  there  may  be  but  slight  leucocytosis,  and  this,  taken  in 
conjunction  with  the  patient's  general  condition,  is  not  a  good 
sign,  but  a  bad  one.  In  many  cases  the  total  number  is  within 
the  normal  limits,  but  whether  this  is  the  case,  or  whether  the 
leucocytes  are  slightly  increased,  a  clue  to  the  condition  will  be 
given  by  the  fact  that  the  polynuclears  show  their  usual  increase, 
and  the  glycogen  reaction  is  present  and  often  very  marked. 

The  haemoglobin,  however,  gives  much  more  valuable  indica- 
tions ;  the  severer  the  case  the  more  rapidly  it  falls,  and  vice  versa. 
The  same  information  can  be  obtained,  though  not  so  well,  from 


DIAGNOSTIC   APPLICATIONS   OF   THE    BLOOD-COUNT     223 

the  variation  in  the  red  corpuscles.  Thus,  in  a  case  of  severe 
sepsis  of  any  sort  the  red  corpuscles  and  haemoglobin  may  fall 
enormously  in  a  few  days.  This  is  a  very  valuable  test  for 
puerperal  fever.  Under  ordinary  circumstances  the  amount  oT 
blood  lost  at  parturition  should  not  lower  the  corpuscles  below 
4,000.000,  and  if  a  count  greatly  below  this  is  found  in  a  few  days' 
time,  there  having  been  no  severe  haemorrhage  in  the  meantime, 
the  diagnosis  of  septic  infection  is  probable,  whatever  be  the 
numbers  of  the  leucocytes ;  if  at  a  later  date  the  numbers  are 
lower  still,  the  diagnosis  is  almost  certain.  The  haemoglobin 
may  fall  10  per  cent,  per  day,  or  even  more  in  a  severe 
case. 

Conversely,  a  rise  in  the  number  of  red  corpuscles  and  haemo- 
globin is  a  good  sign  when  the  existence  of  septic  infection  is 
certain,  whatever  be  the  clinical  condition ;  a  cessation  of  the  fall 
is  good  also,  but  to  a  less  extent.  (Beware,  however,  of  mistaking 
a  concentration  of  the  blood  from  diarrhoea  or  profuse  vomiting 
for  a  true  rise.)  As  an  example  I  may  quote  the  case  of  a  patient 
under  Dr.  Hayes  in  King's  College  Hospital,  in  whom  about  a 
fortnight  after  parturition  there  were  1,306,000  reds,  26  per  cent, 
haemoglobin,  13,400  leucocytes,  of  which  87  per  cent,  were 
polynuclears.  Here  2,700,000  reds  had  been  lost  in  two  weeks, 
indicating  a  very  severe  sepsis.  Her  condition  appeared  desperate, 
yet  in  a  week  the  reds  had  increased  to  2,750,000,  in  a  fortnight 
more  to  3,760,000,  when  for  the  first  time  she  showed  clinical 
improvement.  In  nine  days  more  they  had  reached  4,000,000, 
and  the  patient  was  out  of  danger.  Here  a  good  prognosis  was 
given  solely  on  the  blood-count. 

In  these  cases  a  great  diminution  or  total  disappearance  of  the 
eosinophiles  is  a  bad  sign,  their  reappearance  a  good  one. 

2.  Suppuration. — Where  the  sepsis  is  localized  and  not  so  severe, 
so  that  a  limited  focus  of  suppuration  occurs,  there  is  a  leucocy- 
tosis  which,  in  round  figures,  exceeds  20,000,  and  the  increase  is 
mainly  due  to  a  rise  in  the  polynuclears.  Various  writers  give 
different  figures  as  that  above  which  pus  is  indicated :  some  take 
15,000,  which  I  find  to  be  reached  fairly  often  when  there  is  no 
pus  ;  others  25,000,  which  is  frequently  not  reached  when  sup- 
puration has  occurred.  The  number  20,000  has  been  taken  as 
the  result  of  a  considerable  amount  of  experience  of  all  forms  of 
suppuration,  and  will  prove  a  correct  indication  in  at  least  90  per 
cent,  of  all  cases.     It  is  especially  useful  in  appendicitis,  in  which 


224  CLINICAL   BACTERIOLOGY   AND    HEMATOLOGY 

it  is  an  almost  certain  guide.     The  glycogen  reaction  is  usually 
present,  and  is  a  valuable  confirmatory  test. 

The  following  considerations  must  be  remembered : 

(a)  The  increase  of  leucocytes  to  the  figures  mentioned  above 
only  occurs  when  the  pus  is  pent  up,  not  when  it  occurs  on  a 
free  surface  and  can  escape.  I  have  twice  seen  a  rapid  fall  of 
the  leucocytes  due  to  rupture  of  an  appendicitic  abscess  into  the 
intestine. 

(b)  Where  the  spread  of  the  suppuration  ceases  (due  to  the  death 
or  latency  of  the  organisms  it  contains)  the  leucocytosis  gradually 
subsides,  and  an  old,  thick-walled  collection  of  pus  in  the  tissues 
may  give  a  normal  count.  This  is  especially  common  in  gonor- 
rhoea! pyosalpinx ;  the  gonococcus  dies  out  rapidly,  but  the  pus 
remains,  and  unless  you  examine  the  case  early  there  will  be  no 
leucocytosis,  or  but  little. 

(c)  The  leucocytosis  gives  no  indication  of  the  seat  of  the  pus ; 
there  may  be  a  small  abscess  in  the  body  far  from  the  region 
under  suspicion. 

(d)  The  height  of  the  leucocytosis  gives  no  indication  of  the 
size  of  the  abscess  or  of  its  severity. 

(e)  In  interpreting  a  high  leucocytosis  to  mean  pus,  you  must 
exclude  the  other  causes  of  a  similar  blood  condition — pneumonia, 
etc.  Thus,  in  a  severe  abdominal  disease  leucocytosis  may  be 
due  to  an  ovarian  cyst  with  a  twisted  pedicle,  a  ruptured  tubal 
gestation,  etc. 

(/)  Cold  (tuberculous)  abscesses  do  not  usually  give  a  poly- 
nuclear  leucocytosis;  if  there  is  one,  it  indicates  a  secondary 
septic  infection,  and  is  a  decidedly  bad  sign. 

3.  In  moderate  cases  of  septic  or  other  form  of  inflammation,  sup- 
puration of  free  surfaces,  etc.,  there  is  usually  a  moderate 
leucocytosis  (up  to  18,000),  with  increase  of  the  polynuclears. 

Typhoid  Fever. — In  the  early  stages  there  is  usually  some 
concentration  of  the  blood,  the  red  corpuscles  often  exceeding 
6,000,000 ;  at  a  later  period  they  fall  somewhat,  but  rarely  below 
4,000,000.  In  most  other  diseases  (malaria,  septicaemia,  tubercu- 
losis, etc.)  for  which  typhoid  is  likely  to  be  mistaken  the  rule  is  to 
find  marked  diminution  of  the  red  corpuscles;  when  these  are  high 
with  a  fever  of  some  duration  typhoid  fever  should  be  suspected. 

The  leucocytes  are  usually  normal  or  diminished  in  numbers 
(3,000  to  6,000),  and  there  is  often  slight  excess  of  lymphocytes 
(average  about  50  per  cent.).     This  is  not  unlike  what  occurs  in 


DIAGNOSTIC   APPLICATIONS   OF  THE    BLOOD-COUNT     225 

tubercle  and  malaria,  but  is  of  diagnostic  value  in  distinguishing 
typhoid  from  septicaemia,  in  which  case  there  may  be  no  excess 
of  leucocytes,  but  there  is  usually  a  relative  excess  of  polynuclears. 
The  number  of  leucocytes  in  typhoid  is  of  some  value  in  prog- 
nosis ;  the  lower  the  count,  the  more  severe  the  case,  though  to 
this  rule,  as  to  all  others  in  blood-work,  there  are  exceptions. 

In  the  later  stages  of  the  disease  the  diagnosis  is  best  made  by 
Widal's  reaction. 

Complications. — Here  the  condition  of  the  patient  must  be  taken 
into  account  ;  a  complication  {e.g.,  perforation)  which  causes  a 
rapid  and  marked  leucocytosis  in  a  patient  who  has  not  been 
greatly  enfeebled  by  a  long  and  severe  illness  may  cause  no 
increase,  or  even  a  diminution,  in  an  exhausted  subject.  If  this 
rule  is  forgotten,  the  indications  from  the  blood-count  may  be 
unjustly  stigmatized  as  misleading. 

Perforation, — There  is  a  rapid  increase  of  leucocytes,  which  is 
said  to  occur  in  half  an  hour  ;  the  total  number  may  be  15,000  or 
more.  In  a  patient  who  is  not  greatly  exhausted  this  is  a  very 
sure  sign  ;  exceptions  do  occur,  but  are  not  frequent. 

HcEmorrhage. — If  a  count  has  been  made  a  short  time  previously, 
a  fall  in  the  red  corpuscles  may  be  noticed  in  some  cases,  but  does 
not  always  seem  to  occur.  There  may  also  be  slight  leucocytosis; 
this  is  only  of  importance  in  that  it  leaves  the  diagnosis  of 
perforation  or  haemorrhage  doubtful,  though  raising  the  pre- 
sumption that  one  or  other  has  occurred.  As  a  rule  it  is  not 
high,  and  the  more  it  rises  above  15,000,  the  more  likely  is 
perforation  to  be  the  cause. 

Pneumonia. — When  it  occurs  early  in  the  disease  there  is  usually 
a  slight  leucocytosis ;  when  it  occurs  later  there  may  be  none. 
Do  not  exclude  pneumonia,  therefore,  because  of  its  absence. 

It  follows  from  the  above  that  a  raised  leucocyte  count  in 
typhoid  fever  always  points  to  a  complication  of  some  sort,  but 
does  not  necessarily  indicate  its  nature. 

Pneumonia. — Here  the  results  of  blood  examinations  are 
fairly  constant,  and  of  much  value  in  diagnosis  and  prognosis. 
In  ordinary  cases  there  is  marked  leucocytosis,  due  entirely  to 
an  increase  of  the  polynuclears,  which  may  reach  95  per  cent. 
There  is  also  moderate  secondary  anaemia. 

According  to  Ewing,  who  has  had  much  experience  on  the 
subject,  the  grade  of  leucocytosis  is  roughly  proportionate  to  the 
extent  of  the  lesion.     The  following  are  his  averages : 

15 


226  CLINICAL    BACTERIOLOGY   AND    HEMATOLOGY 

Average  Leucocytes. 

1  lobe  affected         .        .        _        .  20,000 

2  lobes     ,,  ....  22,700 

3  »         n  -         .         -         .  25,000 

4  „         „  ....  27,000 

But  higher  counts,  often  much  higher,  are  frequently  seen. 

These  figures  serve  to  exclude  typhoid  fever,  tuberculosis, 
acute  tuberculous  pneumonia,  and  influenza,  which  do  not  cause 
leucocytosis  ;  a  true  lobar  pneumonia  implanted  on  the  latter 
raises  the  leucocyte  count,  a  lobular  one  does  not. 

In  a  few  cases  of  pneumonia  there  is  no  leucocytosis,  but  these 
hardly  detract  from  the  value  of  the  sign.  They  are  (i)  very 
mild  cases,  and  (2)  very  severe  ones,  usually  rapidly  fatal,  in 
which  the  system  fails  to  react  to  the  infection ;  the  iodine 
reaction  of  the  leucocytes  is  well  marked  in  these.  A  low  count 
in  pneumonia,  therefore,  may  be  a  good  or  bad  sign  :  which  it  is 
can  be  told  by  a  glance  at  the  patient.  A  moderate  leucocytosis 
which  gradually  declines  is  a  bad  sign  in  a  case  of  any  severity. 

As  a  rule  the  leucocytes  fall  to  nearly  normal  at  the  crisis, 
sometimes  a  little  before  ;  in  such  cases  the  crisis  may  be  pre- 
dicted, and  a  sudden  fall  to  normal  after  a  week  or  so  is  of 
very  good  omen.  If  the  leucocytes  remain  up  after  a  crisis  it  is 
most  likely  to  be  due  to  empyema. 

Malaria. — Here,  of  course,  the  diagnosis  should  be  made  by 
finding  the  specific  micro-organism  in  the  blood  (p.  142). 
Where  this  cannot  be  done  the  case  may  still  be  one  of  malaria, 
and  the  blood-count  may  aid  in  the  diagnosis.  There  is  anaemia, 
often  coming  on  rapidly  and  attaining  very  low  figures.  There 
is  no  leucocytosis,  and  according  to  many  observers  there  is  a 
great  increase  in  the  large  lymphocytes,  which  almost  always 
become  more  numerous  than  the  small  ones.  This  test  is  not 
interfered  with  by  the  administration  of  quinine,  which  renders 
the  parasites  difficult  or  impossible  to  find. 

Scarlet  Fever  and  Measles. — In  the  former  there  is,  except 
in  the  very  mildest  cases,  a  marked  leucocytosis  ;  in  the  latter  the 
blood  is  normal  in  the  absence  of  pneumonia  or  other  complica- 
tions. In  scarlet  fever  the  leucocytes  range  from  10,000  to  40,000, 
and  according  to  some  authors  the  prognosis  is  very  bad  in  cases 
showing  more  than  30,000  ;  there  is  an  excess  of  polynuclears 
(80  to  90  per  cent.),  which  is  very  noticeable  in  children,  where 
there  is  usually  a  high  proportion  of  lymphocytes. 


DIAGNOSTIC   APPLICATIONS   OF  THE    BLOOD-COUNT     227 

German  Measles  is  not  accompanied  by  leucocytosis. 

Hooping-cough. — There  is  a  high  grade  of  leucocytosis  (20,000 
to  60,000),  due  mainly  to  an  increase  of  lymphocytes.  This  is  said 
to  occur  before  the  hooping  occurs  and  to  be  of  diagnostic  value, 
but  leucocytosis  with  lymphocytosis  is  so  common  in  children 
that  little  value  should  be  attached  to  it  unless  really  high  figures 
are  found. 

Influenza. — Here  the  blood-count  may  be  of  value,  since,  in 
contradistinction  to  the  majority  of  acute  febrile  diseases,  there 
is  no  leucocytosis  if  complications  are  absent.  In  other  febrile 
diseases  of  rapid  onset — pneumonia,  tonsillitis,  rheumatic  fever, 
septic  affections,  plague,  etc. — leucocytosis  is  almost  constant. 

Rheumatism. — Except  in  the  very  mildest  cases  there  is  leuco- 
cytosis, and,  according  to  Turk,  Ewing,  and  others,  when  there 
are  more  than  20,000  there  is  almost  certainly  some  complication, 
such  as  endocarditis,  pericarditis,  pneumonia,  or  hyperpyrexia.  I 
believe  this  may  be  taken  as  a  safe  general  rule,  though  excep- 
tions do  occur. 

Turk  believes  that  a  clue  to  prognosis  may  be  got  from  the 
percentages  of  eosinophiles  present ;  with  a  proportion  above  the 
normal  the  case  is  likely  to  be  a  mild  one. 

Tuberculosis. — There  is  usually  marked  anaemia  of  the 
secondary  type,  but  in  cases  with  sweating  and  diarrhoea  this 
may  be  masked  by  the  concentration  of  the  blood  ;  an  apparent 
improvement  in  this  respect  may  in  reality  be  a  bad  sign.  The 
leucocytes  are  usually  normal,  though  the  lymphocytes  may  be 
rather  high. 

Where  secondary  septic  infection  takes  place — e.g.^  in  a  vomica 
— the  blood  is  that  of  sepsis  ;  there  is  a  variable  leucocytosis, 
excess  of  polynuclears,  and  advancing  anaemia. 

The  blood-count  is  not  of  much  value  in  the  diagnosis  of 
tubercle ;  it  is  of  some  value  in  prognosis.  Increase  in  the  poly- 
nuclears and  in  the  grade  of  anaemia  are  bad  signs  in  phthisis. 

There  are  one  or  two  precautions  to  be  noted  in  special  cases. 
In  tuberculous  empyema  there  is  frequently  a  secondary  infection, 
and  the  presence  of  a  leucocytosis  does  not  show  that  the  disease 
is  not  tuberculous ;  the  same  thing  applies  to  tuberculous  abscesses 
in  other  parts,  including  the  joints. 

Tuberculous  meningitis  appears  to  offer  the  most  marked 
exception  to  the  rule,  that  uncomplicated  tubercle  does  not  cause 
leucocytosis.      Here  there  is   often   moderate  leucocytosis,  and 

15—2 


228  CLINICAL   BACTERIOLOGY   AND    HEMATOLOGY 

according  to  Horder  it  may  reach  25,000.  I  have  several  times 
met  with  20,000  or  thereabouts.  Tuberculous  pleurisy  is  usually 
without  marked  leucocytosis,  but  occasionally  the  figures  may  reach 
18,000  to  20,000. 

Syphilis. — There  is  usually  progressive  secondary  anaemia  with 
moderate  leucocytosis  (12,000  to  16,000),  due  mainly  to  increase  of 
lymphocytes,  often  of  the  large  type,  but  the  figures  are  too  incon- 
stant to  be  of  much  value  in  diagnosis  (see  p.  180,  Justus's  test). 

Purpura  Hemorrhagica.  —  There  is  naturally  advancing 
anaemia,  which  may  be  associated  with  a  low  or  normal  colour- 
index.  In  the  most  common  type  of  case,  in  which  the  prognosis 
is  relatively  good,  there  is  also  the  usual  slight  polynuclear  leuco- 
cytosis met  with  in  secondary  anaemia,  or  it  may  reach  a  very 
high  grade.  In  some  cases,  however,  in  which  the  prognosis  is 
extremely  bad,  there  is  leucopenia  with  lymphocytosis.  In  one 
case  (under  Dr.  Dalton  at  King's  College  Hospital)  which  was 
fatal  in  a  few  days  the  leucocytes  were  2,000  and  the  lymphocytes 
over  95  per  cent.  A  blood  -  count  should  always  be  made  in 
purpura  haemorrhagica,  when  these  cases  (probably  quite  different 
in  nature)  may  be  diagnosed  early  and  their  gravity  recognised.* 

Malignant  Tumours. — These  are  frequently  associated  with  a 
moderate  leucocytosis,  with  increase  of  the  polynuclears,  and  slight 
anaemia.  The  leucocytosis  is  said  to  be  more  marked  in  the  sar- 
comata than  in  the  carcinomata.  These  facts  are  very  rarely  of 
value  in  diagnosis,  since  whilst  the  growth  is  small  and  removable 
the  blood  is  usually  normal,  except  in  an  ulcerated  carcinoma  of 
the  gastro-intestinal  tract,  and  in  any  case  there  are  so  many 
examples  of  malignant  growth  with  normal  blood,  and  so  many 
causes  of  slight  leucocytosis,  that  its  presence  is  not  much  help. 
Malignant  tumours  of  the  oesophagus  are  occasionally  unaccom- 
panied by  leucocytosis,  but  most  follow  the  general  rule.  > 

Cancer  of  the  Stomach. — The  frequency  with  which  difficulty 
arises  in  the  early  diagnosis  of  this  condition  renders  any  assist- 
ance important,  and  although  the  indications  given  by  the  blood- 
count  are  not  conclusive,  they  are  helpful  in  conjunction  with  the 
clinical  examination  and  the  investigation  of  the  vomit  (p.  115). 
In  some  cases  the  blood  is  normal,  but  usually  there  is  one  of 
two  conditions  :  either  a  marked  secondary  anaemia  with  a  rather 
low  colour-index  (averaging  0-63  according  to  Osier  and  McCrae), 

*  Some  at  least  of  these  cases  are  really  acute  lymphatic  leucocythaemia 
without  glandular  lesions. 


DIAGNOSTIC   APPLICATIONS   OF  THE    BLOOD-COUNT      22g 

with  slight  polynuclear  leucocytosis — about  12,000  to  18,000,  of 
which  80  to  90  per  cent,  are  polynuclears  ;  or  a  condition  closely 
resembling  pernicious  anaemia,  with  a  high  colour-index,  megalo- 
cytes,  and  occasionally  megaloblasts.  In  the  latter  case  the_ 
diagnosis  from  true  pernicious  anaemia  may  usually  be  made  by 
the  fact  that  in  carcinoma  ventriculi  there  is  polynuclear  leucocy- 
tosis instead  of  the  leucopenia  with  lymphocytosis  of  pernicious 
anaemia.     The  blood  finding  in  this  case  is  very  suggestive. 

According  to  some  authors  there  is  no  digestion  leucocytosis  in 
cancer  of  the  stomach,  whilst  there  is  in  other  diseases.  There 
appear  to  be  numerous  exceptions  to  this  rule,  though  it  is  true 
in  the  majority  of  cases,  and  might  be  allowed  some  weight  in 
forming  a  diagnosis.  To  test  for  it  enumerate  the  leucocytes  in 
a  patient  who  has  eaten  nothing  since  the  previous  day ;  let  him 
take  a  meal  (of  which  meat  should  form  part),  and  repeat  the 
examination  in  three  or  four  hours'  time  ;  a  rise  of  2,500  to  3,500 
may  be  considered  normal. 

Ulcer  of  the  Stomach  with  haemorrhage  leads  to  secondary 
anaemia,  but,  unlike  carcinoma  of  the  stomach,  is  usually  un- 
associated  with  leucocytosis.  There  are  exceptions  to  this  rule, 
and  when  the  ulcer  reaches  the  peritoneum  and  causes  local 
peritonitis,  or  ruptures  and  causes  general  peritonitis  or  localized 
abscess,  leucocytosis  occurs. 

Cirrhosis  OF  the  Liver. — In  ordinary  uncomplicated  alcoholic 
cirrhosis  there  may  be  anaemia,  but  there  is  no  leucocytosis,  or  at 
most  very  little.  In  most  of  the  diseases  for  which  it  may  be 
mistaken  an  ordinary  polynuclear  leucocytosis  is  present. 

Hanot's  cirrhosis  is  said  to  be  accompanied  by  leucocytosis, 
but,  as  this  may  be  intermittent,  the  diagnostic  value  of  the  test 
cannot  be  great  in  cases  where  no  leucocytosis  is  found. 

Abscess  of  the  Liver  and  Hydatid  Cyst. — The  former  disease 
is  usually  associated  with  leucocytosis,  the  latter  is  not,  though  here, 
again,  there  are  exceptions.  Some  importance  should  be  attached 
to  the  simple  count,  but  more  to  the  differential  count,  since  in 
hydatid  the  eosinophiles  are  usually  raised  (57  per  cent,  has  been 
recorded,  but  6  to  8  per  cent,  is  more  usual),  and  in  ordinary 
septic  disease  are  absent,  low,  or  normal,  and  these  rules  apply 
w^hether  the  leucocytes  are  normal  or  increased. 

Peritonitis  and  Appendicitis. — See  p.  222,  Sepsis  and  Sup- 
puration. 

Pleurisy  and  Empyema. — With  a  mechanical  pleural  effusion 


230  CLINICAL    BACTERIOLOGY   AND    HEMATOLOGY 

(cardiac  or  renal)  there  is  no  leucocytosis.  With  simple  non-tuber- 
culous pleurisy  the  blood  is  usually  normal,  and  the  same  holds 
in  the  tuberculous  cases,  but  here  the  leucocytes  are  occasionally 
raised  to  18,000  or  more.  Such  cases  may  be  distinguished  from 
pneumonia  or  empyema  by  the  presence  of  the  iodine  reaction  in 
the  latter  and  its  absence  in  pleurisy.  Empyemata  are  always 
associated  with  a  high  leucocytosis,  except  sometimes  in  the 
tuberculous  forms. 

Asthma. — True  asthma  has  a  very  characteristic  blood  condition. 
During  an  attack  there  is  a  leucocytosis  of  moderate  grade,  with 
a  great  increase  of  eosinophiles  ;  10  per  cent,  may  be  taken  as  an 
average,  though  much  higher  counts  have  been  recorded.  In  the 
intervals  the  total  numbers  are  normal,  but  there  is  usually  a 
moderate  eosinophilia,  5  to  7  per  cent,  or  more,  and  I  have  found 
a  slight  increase  of  mast  cells  (about  i  per  cent.),  which  is  so 
rare  a  phenomenon  that  it  may  be  of  some  diagnostic  value. 
Eosinophilia  rarely  occurs  associated  with  other  forms  of  spas- 
modic dyspnoea,  and  its  presence  serves  to  diagnose  asthma  from 
cardiac  or  renal  dyspnoea — often  a  matter  of  great  importance — 
or  from  dyspnoea  due  to  pressure  on  the  bronchi,  trachea,  etc. 

The  sputum  in  asthma  is  usually  characteristic,  and  contains 
the  peculiar  spirals  and  vast  numbers  of  eosinophile  cells  ;  these 
are  rarely  seen  in  cases  of  bronchitis,  but  never  in  any  numbers 
in  other  diseases. 

Bronchitis  and  Broncho-pneumonia. — With  simple  bronchitis 
there  is  the  usual  inflammatory  leucocytosis,  usually  about  12,000 
to  14,000.  In  broncho-pneumonia  the  count  is  much  higher — 
20,000  or  more.     This  applies  to  children  as  well  as  to  adults. 

Endocarditis. — Not  much  help  can  be  obtained  from  the 
leucocytes  in  the  diagnosis  between  the  simple  and  the  malignant 
form,  since  in  either  case  there  may  be  a  normal  or  slightly  raised 
count.  But  in  malignant  endocarditis  there  is  usually  a  rapidly 
increasing  anaemia  of  secondary  type.  The  true  test,  however,  is 
the  bacteriological  one ;  the  blood  is  sterile  in  simple  endocarditis, 
whereas  organisms  are  usually  found  in  ulcerative  cases,  though 
more  than  one  examination  may  be  necessary. 

Valvular  Lesions  require  brief  mention,  since  their  presence 
causes  alterations  which  might  cause  embarrassment  in  the  diag- 
nosis of  other  conditions  if  unrecognised.  With  mitral  lesions,  if 
not  fully  complicated,  there  is  a  tendency  for  an  increase  in  the 
red  corpuscles  (due  to  venous  stasis)  which  may  reach  8,000,000. 


DIAGNOSTIC    APPLICATIONS    OF   THE    BLOOD-COUNT      23X 

In  morbus  caeruleus  it  may  be  still  higher — 10,000,000  or  more. 
In  aortic  disease,  on  the  other  hand,  there  is  a  tendency  to  slight 
anaemia.     The  leucocytes  remain  normal  in  both  cases. 

Puerperal  Fever. — The  great  difficulty  in  interpreting  blood 
findings  in  the  puerperium  is  the  fact  that  the  blood  is  not  normaF 
immediately  before  child-birth.  The  change  affects  the  leucocytes, 
which  are  increased,  it  may  be  as  high  as  36,000  (Cabot) ;  this  is  a 
very  unusual  figure,  and  on  the  average  the  numbers  do  not  exceed 
16,000  in  primiparae  and  12,000  in  multiparae.  Leucocytoses  within 
these  limits  are  to  be  looked  on  as  being  probably  normal,  and  not 
as  indicating  sepsis.  More  information  may  be  gained  by  counts 
at  intervals.  The  figures  should  decrease  rapidly  after  delivery, 
becoming  normal  in  less  than  a  fortnight,  and  if  a  count  remains 
the  same  on  two  successive  days  an  inflammatory  process  is 
suggested,  and  a  definite  rise  constitutes  almost  absolute  proof. 
It  is  in  these  cases  especially  that  the  glycogen  reaction  is  of 
value,  and  its  presence  should  outweigh  that  of  the  total  numbers. 
The  differential  count  is  not  of  much  value,  since  the  polynuclears 
are  increased  in  normal  pregnancy,  unless  these  cells  reach  90  per 
cent,  or  more. 

The  chief  reliance  is  to  be  placed  on  the  haemoglobin.  It 
should  be  normal  or  slightly  reduced  at  the  end  of  pregnancy, 
fall  in  proportion  to  the  haemorrhage  at  parturition  and  for  a  day 
or  two  after,  and  then  be  rapidly  regenerated.  Under  normal 
circumstances  it  should  not  be  much  below  70  per  cent,  nor  the 
red  corpuscles  much  below  4,000,000.  Figures  much  below  these 
(unless  there  has  been  great  haemorrhage,  or  unless  it  has  been 
repeated)  raise  suspicions  of  sepsis,  whilst  an  observed  fall  is 
almost  definite  proof.  This  has  also  much  value  in  prognosis 
(see  p.  223). 

Perimetritis,  Parametritis,  etc. — Here  the  usual  relations 
hold  good.  There  is  a  moderate  leucocytosis  in  a  non-suppurative 
lesion,  a  high  one  when  suppuration  occurs.  The  figures  are 
usually  somewhat  lower  than  in  other  parts  of  the  body,  and 
T  8,000  may  be  taken  as  fairly  definite  evidence  of  pus,  provided 
other  sources  of  leucocytosis  can  be  excluded. 

Other  Pelvic  Swellings. — The  blood-counts  in  these  cases 
have  to  be  interpreted  with  much  caution,  and  are  often  very 
equivocal.  Thus,  pyosalpinx  is  usually  associated  with  the  ordinary 
signs  of  pus,  but  the  cases  are  frequently  tuberculous,  when  there 
is  no  leucocytosis,  or  gonorrhceal,  when  there  is  only  leucocytosis 


232  CLINICAL    BACTERIOLOGY   AND    HEMATOLOGY 

if  the  count  is  made  whilst  suppuration  is  in  progress.  The  sterile 
collections  of  pus  left  after  an  attack  of  gonorrhceal  salpingitis 
do  not  cause  leucocytosis.  A  normal  count,  therefore,  does  not 
exclude  pus  in  the  tubes.  Similarly,  there  are  exceptions  to  the 
rule  that  simple  ovarian  tumours  and  cysts  are  associated  with 
normal  blood.  Where  there  is  inflammation  and  formation  of 
adhesions  there  is  slight  leucocytosis,  where  there  is  much  peri- 
toneal irritation  a  higher  one,  and  with  twisting  of  the  pedicle 
very  high  figures  may  be  reached.  These  latter  are  liable  to  be 
mistaken  for  acute  peritonitis,  but  the  leucocytes  do  not  give  the 
iodine  reaction.  Further,  malignant  ovarian  tumours  usually  cause 
a  slight  leucocytosis  and  decrease  of  reds  ;  this  has  been  suggested 
as  of  diagnostic  importance,  but  it  might  be  due  to  so  many  other 
conditions  that  not  much  reliance  can  be  placed  on  it.  A  normal 
condition  of  the  blood,  however,  would  be  some  evidence  against 
malignancy.  In  tubal  gestation  the  blood  is  normal.  After  rupture 
there  is  anaemia  and  increase  of  the  leucocytes,  which  may  reach 
24,000  or  more. 

Pemphigus,  Dermatitis  Herpetiformis,  and  Erythema 
Multiforme,  are  associated  with  a  high  percentage  of  eosino- 
philes,  and  this  is  of  importance  in  the  diagnosis  of  these  diseases 
from  local  infective  processes,  which  they  often  closely  resemble. 
The  eosinophiles  in  the  former  group  of  diseases  may  be  expected 
to  exceed  10  per  cent.,  and  may  be  much  higher,  and  there  may 
be  a  high  leucocytosis. 


PART  III 

CYTO-DIAGNOSIS 

Cyto-diagnosis  is  the  diagnosis  of  the  cause  of  exudates  by  the 
recognition  of  the  cells  which  they  contain.  It  may  be  regarded 
as  a  branch  of  haematology,  though  the  cells  which  are 
encountered  are  not  wholly  those  of  the  blood.  Its  results  are 
less  certain  than  those  obtained  by  the  recognition  of  the  organism 
(if  any)  present  in  the  exudate,  but  are  often  easier  to  obtain  ; 
they  are  more  certain  than  those  obtained  by  an  examination  of 
the  blood,  as  easy  to  obtain,  and  in  most  cases  very  much  easier 
to  interpret.  As  the  methods  are  very  simple  and  require  no 
special  apparatus  (though  a  centrifuge  is  a  very  great  help),  they 
are  within  the  reach  of  all  practitioners,  and  a  cytological  examina- 
tion should  always  be  made  when  fluid  is  withdrawn  from  the 
chest,  abdomen,  etc.,  whether  for  diagnosis  or  treatment. 

Method  of  collecting  the  Cells. — No  description  will  be 
given  of  the  methods  of  obtaining  the  exudate,  as  those  which 
are  not  in  general  use  have  been  dealt  with  already. 

If  the  fluid  does  not  clot  spontaneously,  it  is  only  necessary  to 
centrifugalize  a  portion  (as  much  as  the  tube  will  hold — about 
lo  c.c.)  for  five  minutes  or  so,  and  then  to  invert  the  tube  and  pour  off" 
as  much  of  the  supernatant  fluid  as  will  come  away.  The  sediment 
will  be  left,  and  a  drop  or  two  of  fluid  will  run  back  down  the 
sides  of  the  tube.  These  must  be  thoroughly  mixed  in  with  the 
deposit  so  as  to  form  a  uniform  emulsion. 

In  the  absence  of  a  centrifugal  machine,  allow  the  fluid  to 
stand  for  twelve  hours  or  so  to  settle,  adding  a  crystal  of  thymol 
to  prevent  decomposition.  Then  remove  some  of  the  deposit 
with  a  pipette ;  you  cannot  invert  the  tube  in  this  case,  as  the 
sediment  is  not  so  compact,  and  will  pour  out. 

233 


234  CLINICAL    BACTERIOLOGY   AND    HEMATOLOGY 

When  the  fluid  has  coagulated,  put  it  in  a  strong  bottle  with 
some  glass  beads  or  balls,  or  fragments  of  glass  of  any  sort,  and 
shake  for  ten  minutes.  This  will  break  up  the  coagulum  and  set 
most  of  the  cells  free.  Allow  the  fluid  to  stand  for  a  minute  or 
two  so  that  the  pieces  of  fibrin  may  settle  ;  then  decant  the 
supernatant  fluid  into  the  centrifugalizing  or  sedimenting  tube, 
and  proceed  as  before. 

Where  the  fluid  is  pus  no  preparation  is  usually  necessary.  If 
it  clots  it  does  so  very  feebly,  and  in  this  case  a  little  stirring 
with  a  platinum  loop  will  set  free  plenty  of  cells  for  examination, 
or  you  may  take  up  the  clot  with  a  loop  and  rub  it  on  the  slide  or 
cover-glass. 

Method  of  preparing  the  Specimen  for  Examination. — 
The  specimens  may  be  examined  wet  or  dry.  In  most  cases  the 
former  method  is  best,  as  it  is  quicker,  and  often  yields  information 
which  cannot  be  obtained  by  a  dry  specimen.  The  preparations, 
however,  do  not  keep,  and  where  permanent  ones  are  required 
the  method  is  inapplicable. 

Wet  Method. — Place  one  drop  of  watery  methylene  blue  or 
borax  methylene  blue  on  a  slide  and  add  two  or  three  drops  of 
the  emulsion  of  cells.  Stir  with  the  platinum  loop  or  needle, 
allow  the  mixture  to  stand  for  two  or  three  minutes,  and  then 
apply  a  cover-glass.  The  cover  glass  may  be  cemented  to  the 
slide  by  means  of  melted  paraflin  applied  with  a  hot  iron  rod ;  it 
is  best  to  do  this  if  the  oil-immersion  lens  is  to  be  used,  otherwise 
the  suction  of  the  lens  may  lift  up  the  cover-glass. 

Or,  put  two  or  three  drops  of  the  emulsion  on  a  slide,  cover, 
and  examine  without  staining.  Then  put  a  drop  or  two  of  acid 
methylene  blue  (see  p.  30)  on  the  slide  just  touching  the  cover- 
glass  ;  it  will  pass  in  by  capillarity,  and  at  different  distances 
from  the  edge  you  will  get  an  unstained  area,  an  area  where  the 
stain  is  faint,  but  very  selective,  and  a  deeply  stained  area.  The 
middle  zone  is  best  to  examine.  The  red  corpuscles,  if  present, 
will  be  dissolved  by  the  acid,  and  after  a  few  minutes  the  cells 
will  be  stained  with  great  distinctness. 

Dry  Method.  —  Prepare  films  on  the  slide  or  cover-glass 
(p.  199),  using  the  emulsion  exactly  as  if  it  were  blood.  This 
may  be  stained  by  Jenner's  method  (p.  204)  or  fixed  and  stained 
subsequently.  Any  of  the  fixing  and  staining  methods  described 
for  blood  may  be  used,  but  I  prefer  to  allow  the  film  to  dry,  fix 
with  saturated  solution  of  perchloride  of  mercury  for  a  minute 


CYTO-DIAGNOSIS  235 

or  two,  wash,  stain  in  carbol  thionin  for  two  minutes,  wash,  dry 
and  mount.  This  renders  everything  very  distinct  except  (some- 
times) the  nucleoli  of  the  endothelial  cells  and  the  granules  of  the 
polynuclears.  Where  the  latter  have  to  be  inquired  into  (which 
rarely  happens)  the  film  must  be  fixed  with  heat  and  stained  with 
triacid  (p.  204). 

Cells  met  with  in  Exudates. 

Leucocytes  derived  from  the  blood  are  present  in  the  majority 
of  exudates,  and  in  most  cases  they  are  of  ordinary  appearance, 
and  readily  recognised  from  the  descriptions  already  given.  The 
polynuclears,  however,  may  undergo  various  forms  of  degeneration, 
and  become  so  altered  that  their  nature  may  be  difficult  to  make 
out.  This  occurs  mainly  in  old  exudates,  especially  in  pus. 
There  are  three  chief  forms  : 

(a)  In  some  cases  the  nucleus  undergoes  fragmentation — i.e.^ 
breaks  up  into  several  isolated  masses,  so  that  the  cell  becomes 
truly  polynuclear  (Plate  IX.,  Fig.  2,  where  the  ingested  leuco- 
cytes are  fragmented).  These  masses  stain  deeply,  and  the  cell 
is  easy  to  recognise,  but  it  often  happens  that  the  fragments 
of  the  nuclei  are  set  free,  and  may  then  be  mistaken  for  small 
lymphocytes.  If  a  specimen  be  stained  by  the  triacid  stain,  they 
may  often  be  distinguished  by  a  few  granules  which  remain 
adherent  to  the  nucleus. 

(b)  Dropsy  of  the  nucleus,  which  converts  it  into  a  large  circular 
or  reniform  mass  which  stains  faintly.  In  this  case  the  cell 
resembles  a  large  lymphocyte,  large  hyaline  leucocyte,  or  myelo- 
cyte. It  may  usually  be  distinguished  from  the  former  by  the 
presence  of  granulations,  and  the  latter  is  not  known  to  occur 
in  exudates  except  in  cases  of  leucocythaemia. 

{c)  Fatty  degeneration  of  the  cell,  shown  by  the  occurrence  of 
clear  refractile  granules  with  sharp  contour.  These  are  only 
seen  in  wet  preparations.  In  this  case  the  cell  usually  undergoes 
severe  degenerative  changes  or  even  complete  solution,  and  in 
old  pus  it  may  be  difficult  to  make  out  any  definite  cells  at  all 
(Plate  IX.,  Fig.  3). 

As  a  matter  of  fact,  these  degenerative  changes  rarely  cause  the 
slightest  difficulty  in  diagnosis.  It  frequently  happens  that  no 
pathologist  is  able  to  say  definitely  what  is  the  nature  of  any 
particular  cell,  but  the  nature  of  the  cells  as  a  whole  is  usually 
obvious  at  a  glance. 


236  CLINICAL   BACTERIOLOGY   AND    HEMATOLOGY 

Red  corpuscles  occur  frequently,  especially  in  tuberculous  and 
malignant  exudates,  which  may  be  definitely  haemorrhagic.  It  is 
necessary  to  distinguish  corpuscles  belonging  to  the  exudate, 
which  are  intimately  mixed  with  it,  from  those  derived  from  the 
puncture,  in  which  case  the  blood  is  most  marked  at  the  beginning 
or  end  of  the  flow. 

Endothelial  cells  are  very  important,  and  it  is  necessary  to  be 
able  to  recognise  them  at  once.  In  certain  passive  exudates 
(due  to  cardiac  and  renal  disease)  these  cells  occur  as  large  flat 
plates,  exactly  as  if  the  endothelial  coat  had  been  scraped  ofi"  the 
pleura  (Plate  X.,  Fig.  2).  They  are  then  seen  to  be  much  larger 
than  the  largest  of  leucocytes,  and  to  have  a  diameter  three  or  four 
times  that  of  a  red  corpuscle.  Each  has  a  nucleus  (sometimes 
more)  which  does  not  usually  stain  very  deeply,  protoplasm  which 
stains  more  faintly  still,  and  one  or  more  nucleoli  which  stain 
very  deeply  in  wet  preparations,  less  so  in  dry  ones. 

These  cells  are  often  grouped  into"  placards"  (Plate  X.,  Fig.  2), 
the  edges  of  adjoining  cells  fitting  into  one  another  like  those  of  the 
counties  on  a  map.  The  groups  of  cells  thus  formed  are  always 
flat,  and  careful  focussing  up  and  down  shows  that  they  consist 
of  a  single  layer  of  cells — an  important  fact,  as  it  distinguishes 
them  from  masses  of  cells  of  a  malignant  growth. 

Endothelial  cells  are  very  phagocytic,  and  ingest  bacteria,  red 
corpuscles  (Plate  X.,  Fig.  3),  leucocytes  (Plate  IX.,  Fig.  2),  etc. 
They  often  undergo  fatty  degeneration  (Plate  X.,  Fig.  i)  or 
general  degeneration,  shown  by  their  very  faint  staining ;  complete 
solution  of  the  protoplasm  may  occur,  and  the  nucleus  be  set  free. 
It  may  then  be  mistaken  for  a  lymphocyte. 

Where  inflammation  takes  place  in  a  serous  membrane  the 
first  thing  that  happens  is  that  the  endothelial  cells  are  set  free, 
so  that  they  are  always  found  with  the  leucocytes  in  the  early 
stages  of  pleurisy  or  peritonitis.  If  the  inflammation  is  severe 
they  are  destroyed,  and  the  fluid  at  a  later  date  does  not  contain 
them.  If  the  inflammation  is  less  intense  they  are  stimulated  to 
growth,  and  the  young  proliferating  forms  are  often  very  similar 
to  the  large  lymphocytes  and  hyaline  leucocytes.  They  vary 
greatly  in  size,  forming  a  continuous  series  between  a  cell  as  large 
as  the  large  lymphocyte  to  one  as  large  as  the  plates  described 
above.  They  are  round  or  oval,  not  mutually  adapted,  as  in  the 
endothelial  cells  which  have  desquamated  in  passive  exudates  ; 
sometimes  two  hemispherical  cells  may  be  found  in  apposition 


CYTO-DIAGNOSIS  237 

(Plate  X.,  Fig.  i).  The  smaller  {i.e. ^  younger)  the  cell,  the  smaller 
is  the  ring  of  protoplasm  in  proportion  to  the  size  of  the  nucleus, 
and  the  more  deeply  does  it  stain.  Cells  of  the  type  described 
above  will  be  referred  to  as  ''  active  "  endothelial  cells,  in  contra- 
distinction to  the  "  passive  "  plaques  of  desquamated  endothelium. 
Malignant  Cells. — These  cannot  be  distinguished  with  certainty 
from  some  types  of  endothelial  cells— at  least,  I  must  confess 
myself  unable  to  do  so.  Cells  in  mitosis  are,  of  course,  very  sug- 
gestive, but  very  rare,  and  there  is  no  reason  why  they  should  not 
occur  in  ordinary  active  endothelium.  But  malignant  cells  may 
occur  grouped  in  a  characteristic  way  (p.  239). 

Pleuritic  Effusions. 

It  is  in  these  that  cyto-diagnosis  is  of  chief  value,  and  its  results 
most  trustworthy.  A  diagnosis  based  on  the  subsequent  rules 
will  rarely  be  found  erroneous. 

Tuberculous  Pleurisy. — Two  forms  are  to  be  recognised : 
the  primary,  the  so-called  idiopathic  form,  in  which  the  prognosis 
is  good  as  regards  immediate  recovery,  but  which  indicates  a 
great  probability  that  the  patient  will  subsequently  become 
phthisical ;  and  the  secondary,  which  is  due  to  the  extension  of  a 
tuberculous  lesion  to  the  surface  of  the  lung,  and  is  probably  due 
to  tubercle  plus  mild  sepsis. 

Primary  Tuber cidotis  Pleurisy. — The  fluid  is  fairly  clear,  yet 
yields  numerous  cells  on  centrifugalization.  It  usually  clots 
spontaneously. 

The  cells  are  almost  all  lymphocytes,  and  there  are  some  red 
blood-corpuscles  (Plate  IX.,  Fig.  i).  There  may  also  be  a 
few  large  endothelial  cells,  flat  plates  with  a  well-marked  nucleus, 
and  often  one  or  more  nucleoli ;  their  characters  will  be  described 
more  fully  subsequently.  They  are  cells  which  have  been  desqua- 
mated from  the  pleura,  and  play  no  part  in  the  pathological  process. 

In  cases  examined  at  an  early  stage  there  may  be  a  few  poly- 
nuclear  cells — up  to  15  per  cent.  As  the  case  progresses,  these 
become  fewer  and  fewer,  and  after  the  first  week  only  isolated 
specimens  can  be  seen.  The  diagnosis  may  be  clinched  in  some 
of  these  cases  by  the  demonstration  of  the  tubercle  bacillus. 

Secondary  Tuhercidous  Pleurisy. — In  this  case  the  lymphocytes  are 
mixed  with  polynuclear  leucocytes  in  approximately  equal  proportions  ; 
this  is  a  reason  for  thinking  that  there  is  a  septic  element  super- 
added to  the  tuberculous  one.     As  the  case  progresses,  the  septic 


238  CLINICAL    BACTERIOLOGY   AND    HEMATOLOGY 

organisms  may  gain  entrance  in  larger  numbers,  and  the  case 
would  become  indistinguishable  from  an  ordinary  empyema  unless 
a  cytological  examination  had  been  made  early;  or,  and  this  is 
more  usual,  the  polynuclears  may  gradually  disappear,  and  the 
case  become  a  simple  tuberculous  one. 

In  tuberculous  pyopneumothorax  the  polynuclears  greatly  pre- 
dominate, but  lymphocytes  are  usually  present  in  fair  numbers. 
The  lesion  is  due  to  the  bursting  of  a  vomica  or  abscess  that  is 
almost  necessarily  septic. 

"Septic"  Exudates  {i.e.^  those  due  to  the  pneumococcus, 
streptococcus,  gonococcus,  and  similar  pyogenic  bacteria). — The 
characteristic  cell  is  the  polynuclear  leucocyte.  In  the  early 
stages  the  films  show  these  cells  in  large  numbers,  and  they  are 
mixed  with  red  corpuscles  and  with  endothelial  cells  of  active 
type  (Plate  IX.,  Fig.  2).  The  pathogenic  organism  may  be 
distinguished  either  in  films  or  in  cultures.  The  process  may 
evolve  on  one  of  two  lines,  and  in  either  case  the  cytology  is 
fairly  characteristic. 

{a)  The  process  is  mild,  and  recovery  takes  place ;  this  is  most 
likely  to  occur  when  the  inflammation  is  due  to  the  gonococcus 
(in  joints  especially)  or  to  the  pneumococcus  in  a  strong  subject. 
The  polynuclears  and  endothelial  cells  become  more  and  more 
scanty,  and  lymphocytes  make  their  appearance  in  increasing 
numbers.  The  discovery  of  these  cells  in  a  septic  exudate  is  a 
good  sign ;  the  discovery  of  the  pathogenic  organism  mainly  or 
entirely  within  the  cells  is  another. 

(h)  The  process  may  pass  on  to  suppuration.  In  this  case  the 
endothelial  cells  become  less  and  less  abundant,  and  the  poly- 
nuclears become  more  numerous  and  undergo  the  various  forms 
of  degeneration  described  above  (Plate  IX.,  Fig.  3). 

Cryptogenic  Pleurisy,  possibly  due  to  True  Rheumatism. 
— In  this  case  the  predominating  cell  is  the  active  endothelial  cell 
in  various  stages  of  fatty  degeneration,  and  in  addition  there  is  a 
comparatively  small  number  of  all  the  leucocytes  in  approxi- 
mately the  same  proportions  as  in  the  blood,  and  a  few  red 
corpuscles.  The  exudate  is  sterile.  These  appearances  are 
found  in  the  very  rare  cases  of  true  rheumatic  pleurisy,  and  may 
be  regarded  as  a  good  sign,  in  that  they  do  not  indicate  a  tuber- 
culous or  septic  origin  (Plate  X.,  Fig.  i). 

Pleuritic  Exudates  due  to  Malignant  Disease. — The 
appearances   vary,   and   a  definite   diagnosis  cannot  always   be 


CYTO-DIAGNOSIS  239 

made.  There  is  no  criterion  (other  than  certain  mitoses,  which, 
as  far  as  I  know,  have  never  been  made  out  in  an  exudate) 
by  which  an  isolated  mahgnant  cell  can  be  distinguished  from  an 
active  endothelial  cell.  In  some  cases,  however,  the  masses  qL 
cells,  which  can  be  seen  to  be  solid  and  several  cells  thick/''  occur 
in  the  exudate  mixed  with  red  corpuscles  and  a  few  leucocytes. 
These  cells  are  variable  in  size,  usually  stain  deeply,  and  often 
have  a  well-marked  nucleolus.  In  the  figure  shown  (which  comes 
from  the  ascitic  fluid  in  a  case  of  carcinoma  of  the  ovary)  the 
resemblance  to  an  alveolus  of  carcinoma  as  seen  in  a  section  is 
very  distinct  (Fig.  53). 


Fig.  53. — Malignant  Masses  in  Ascitic  Fluid, 

In  other  cases,  and  much  more  frequently,  these  masses  are 
absent,  and  their  place  is  taken  by  large  endothelial  cells  (often 
many  times  larger  than  a  red  corpuscle),  which  can  be  seen  in  a 
wet  preparation  to  be  in  various  stages  of  fatty  and  other  forms 
of  degeneration,  and  which  in  dry  preparations  are  found  to  be 
extensively  vacuolated  (Plate  X.,  Fig.  3),  and  often  contain 
ingested  red  corpuscles.  These  may  be  arranged  in  masses,  are 
mixed  with  red  corpuscles,  and  perhaps  a  leucocyte  or  two. 

Some  writers  consider  these  cells  to  be  growth-cells,  but  on 
what  grounds  I  do  not  know.  When  they  form  masses  they  are 
always  one  cell  thick,  never  solid  alveoli ;  and,  more  conclusive, 

♦  As  shown  by  focussing  up  and  down  whilst  examining  a  wet  specimen,  so 
as  to  obtain  a  series  of  "  optical  sections." 


240  CLINICAL    BACTERIOLOGY   AND    HEMATOLOGY 

when  an  opportunity  is  obtained  of  examining  the  cells  of  the 
growth  post-mortem,  they  are  often  absolutely  different  from 
those  found  in  the  exudate  during  life. 

In  yet  other  cases  the  endothelial  cells  are  of  the  ordinary 
passive  type,  but  a  suspicion  of  the  nature  of  the  growth  may  be 
obtained  from  the  number  of  red  corpuscles  present. 

"  Mechanical  "  Exudates  (i.e.,  those  due  to  cardiac  disease, 
pulmonary  congestion,  or  renal  disease). — The  deposit  from  the 
exudate  is  usually  very  scanty,  and  consists  of  large  flat  masses 
of  passive  endothelial  cells,  there  being  often  many  cells  in 
one  large  plate ;  their  outlines  may  be  indistinct.  There  is 
usually  nothing  else,  but  there  may  be  a  few  red  corpuscles  or 
leucocytes. 

Pleurisy  Secondary  to  Infarcts. — Endothelial  cells  mixed 
with  much  blood  and  with  many  polynuclear  leucocytes  have 
been  described,  but  I  have  no  personal  experience  of  the  condition. 

Peritoneal  Exudates. 

These  are  very  equivocal,  and  often  difficult  or  impossible  to 
interpret.  The  ultimate  conditions  leading  to  the  production  of 
the  cells  are  doubtless  the  same  in  the  peritoneum  as  in  the  pleura, 
but  here  the  fluid  is  in  close  proximity  to  the  intestine,  and  liable 
to  constant  mild  infective  processes.  These  call  forth  a  poly- 
nuclear leucocytosis,  which  is  very  common  in  ascitic  fluid,  and 
devoid  of  the  significance  which  it  has  in  the  pleura. 

Tuberculous  Peritonitis  may  be  accompanied  by  a  pure 
lymphocytosis,  the  cells  becoming  extraordinarily  abundant,  so 
that  the  fluid  may  be  turbid,  or  there  may  be  polynuclears  in  a 
practically  pure  state.  I  do  not  think  the  condition  can  be  diag- 
nosed unless  tubercle  bacilli  are  found. 

Septic  Peritonitis. — The  cells  are  all  polynuclears,  except  in 
the  early  stages,  in  which  a  few  endothelial  cells  and  red  corpuscles 
may  be  found.  The  diagnosis  is  to  be  made  by  the  discovery  of 
the  organism,  which  is  usually  easy. 

Mechanical  Ascites  {i.e.,  that  due  to  cirrhosis  of  the  liver, 
renal  disease,  cardiac  disease,  etc.). — Here  endothelial  cells,  often 
in  masses,  are  almost  always  present,  and  sometimes  practically 
unmixed  with  other  cells.  But  chronic  inflammation  of  the 
peritoneum,  with  or  without  mild  sepsis,  is  frequently  present, 
and  polynuclears  and  lymphocytes  frequently  occur. 


C\TO-DIAGNOSIS  24I 

Malignant  Disease. — Here  the  large  vacuolated  endothelial 
cells  shown  on  Plate  X.,  Fig.  3,  may  occur,  and  are  extremely 
suggestive,  though  they  cannot  be  taken  as  definite  proof.  Rarely 
you  may  find  definite  malignant  masses,  which,  of  course,  settles 
the  diagnosis.  In  other  cases  there  may  be  numerous  polynuclear 
cells,  and  in  yet  others  mostly  lymphocytes.  The  presence  of  an 
abundance  of  red  cells  is  suggestive,  provided  you  can  be  sure  it 
does  not  come  from  the  puncture,  in  which  case  it  will  be  most 
abundant  at  the  beginning  of  the  flow.  It  will  be  apparent  that 
the  diagnosis  of  malignancy  cannot  be  made  in  the  majority  of 
cases  by  the  cytology  of  the  ascitic  fluid. 

The  Meninges. 

Here  the  technique  is  somewhat  different.  Clotting  is  not  so 
likely  to  occur,  and  when  it  does  so  is  much  slower,  so  that  if  the 
fluid  can  be  examined  in  any  reasonable  time  there  is  no  necessity 
to  break  up  the  clot.  This  is  fortunate,  since  the  number  of 
cells  present  is  of  importance,  and  they  cannot  be  counted  in  a 
specimen  which  has  coagulated.  My  own  method  is  to  count  the 
cells  directly,  without  concentration  and  without  dilution,  in  a 
Thoma-Zeiss  counting-chamber,  by  the  method  used  for  the 
leucocytes  and  described  on  p.  193.  If  the  specimen  has  had  time 
to  sediment,  shake  it  thoroughly ;  then  place  a  loopful  or  two  on 
the  counting-chamber,  cover  it,  getting  Newton's  rings,  and  allow 
to  settle.  Then  arrange  the  microscope  so  that  the  diameter  of 
the  field  is  equal  to  that  of  eight  small  squares,  and  proceed  to 
count  the  leucocytes  on  forty  or  eighty  fields ;  in  the  former  case 
the  result  multiplied  by  two  gives  the  number  of  cells  per  cubic 
millimetre,  whilst  if  eighty  are  counted  the  number  is  given 
direct,  no  calculation  being  necessary.  (There  is  no  dilution  of 
the  fluid,  and  you  have  counted  the  actual  number  in  ^  or  i  cubic 
millimetre.)  The  only  difiiculty  arises  if  red  corpuscles  are 
present ;  they  may  be  distinguished  by  being  less  granular  and 
less  refractile  than  the  leucocytes,  and  are  not  to  be  counted. 

The  following  rules  may  be  taken  as  approximately  correct  for 
the  numbers  of  cells  met  with  in  various  conditions.  In  health 
there  may  be  none,  and  never  more  than  single  figures  per  cubic 
millimetre  :  the  average  is  perhaps  one  or  two.  In  "  aseptic " 
meningitis — i.e.,  that  due  to  syphilis,  or  that  which  occurs  in  tabes, 
general  paralysis,  some  forms  of  herpes,  and  in  almost  any  organic 
lesion  involving  the  meninges — the  number  per  cubic  millimetre 

16 


242  CLINICAL    BACTERIOLOGY   AND    HEMATOLOGY 

is  expressed  in  two  or  three  figures,  and  usually  ranges  between 
50  and  200.  In  tuberculous  meningitis  the  numbers  are  higher,  and 
1,000  is  perhaps  the  average,  but  it  may  go  much  higher,  and  in 
**  septic  "  meningitis^  including  cevehvo -spinal  meningitis,  the  numbers 
are  very  large,  often  running  into  tens  or  even  hundreds  of 
thousands. 

Having  counted  the  leucocytes,  proceed  to  centrifugalize  the 
fluid,  and  examine  films  from  the  deposit  by  the  wet  or  dry  method. 
The  former  shows  the  cells  more  clearly,  and  is  to  be  preferred 
when  a  cytological  examination  only  is  required,  as  in  the  diagnosis 
of  tabes  or  general  paralysis.  Where  bacteria  are  to  be  looked 
for,  dried  films  should  be  made  and  stained  by  Jenner's  method, 
or  fixed  with  perchloride  and  stained  by  thionin.  Then  proceed 
with  the  chemical  examination  of  the  fluid  already  described. 

Normal  fluid  occurs  in  any  nervous  disease  not  attended  by  an 
organic  lesion  of  the  meninges  :  deep  cerebral  tumours,  hysteria, 
deep  cerebral  haemorrhages,  peripheral  neuritis,  epilepsy,  syringo- 
myelia, etc. 

In  cerebral  tumour  the  fluid  may  be  under  excessive  pressure,  so 
that  it  squirts  out  of  the  needle  ;  in  such  cases  there  may  be  great 
relief  to  the  headache  after  the  withdrawal  of  a  considerable 
amount  of  fluid.  With  a  cortical  tumour  there  is  usually  slight 
lymphocytosis.  According  to  some  writers,  the  pressure  is 
moderately  raised  in  epilepsy. 

Aseptic  meningitis,  using  the  term  to  indicate  that  there  are  no 
cultivable  organisms  present,  occurs  in  syphilis,  tabes,  general 
paralysis,  superficial  gummata  and  other  tumours,  insular  sclerosis, 
chronic  alcoholic  meningitis,  hypertrophic  pachymeningitis,  acute  softening, 
some  cases  of  herpes,  etc.  In  these  cases  we  may  expect  to 
find  a  hundred  or  two  leucocytes — practically  all  of  which  are 
lymphocytes — per  cubic  millimetre.  In  addition  there  is  usually 
a  slight  excess  of  albumin,  and  sugar  is  present,  though  some- 
times reduced  in  amount. 

A  slight  lymphocytosis,  therefore,  does  not  in  itself  give  a  clue 
to  the  diagnosis  unless  it  rests  between  two  conditions,  one  of 
which  causes  lymphocytosis,  whilst  the  other  does  not.  Thus,  if 
the  diagnosis  is  either  insular  sclerosis  or  hysteria,  the  presence 
of  a  moderate  leucocytosis  tells  strongly  in  favour  of  the  former. 
Similarly  in  the  differential  diagnosis  between  tabes  and  peripheral 
neuritis,  and  between  general  paralysis  and  most  of  the  diseases 
which  it  simulates.    It  is  to  be  noted  that  excess  of  lymphocytes 


CYTO-DIAGNOSIS  243 

in  the  cerebro-spinal  fluid  is  a  very  early  and  a  very  constant  finding 
in  these  affections,  and  often  occurs  long  before  the  diagnosis  can 
be  made  by  ordinary  clinical  methods.  Its  absence  is  most  im- 
portant as  a  negative  test ;  its  presence  is  only  equally  important  . 
if  the  diagnosis  certainly  lies  between  a  disease  in  which  this  sign 
is  present  and  one  in  which  it  is  absent. 

Syphilis. — In  syphilis  without  involvement  of  the  meninges 
the  cerebro-spinal  fluid  remains  normal,  but  with  the  slightest 
involvement  of  these  structures  a  lymphocytosis  occurs. 

Tuberculous  Meningitis  has  been  described  already.  The 
lymphocytes  may  be  so  numerous  as  to  make  the  fluid  very 
slightly  turbid  as  compared  with  pure  water,  and  may  number 
10,000  per  cubic  millimetre.  There  is  a  slight  excess  of 
albumin,  no  sugar,  and  sometimes  albumose  is  present.  Occa- 
sionally the  films  show  a  few  polynuclear  cells,  but  they  are  never 
numerous. 

HEMORRHAGE. — With  a  deep  cerebral  haemorrhage  the  fluid  is 
usually  clear  for  two  or  three  days,  and  then  tinged  with  blood  and 
blood  pigment ;  the  time  necessary  for  this  to  occur  depends  on 
the  depth  of  the  haemorrhage  from  the  surface  or  from  the 
cerebral  ventricles. 

When  you  find  blood  in  the  cerebro-spinal  fluid,  make  sure  that 
it  does  not  come  from  the  parietes.  If  this  is  the  case,  it  will  be 
most  abundant  or  limited  to  the  commencement  of  the  flow,  and 
if  much  is  present  the  fluid  will  coagulate.  If  it  is  due  to  a  lesion 
it  will  be  intimately  mixed  with  the  fluid,  and  will  not  coagulate. 
After  blood  has  been  present  in  the  cerebro-spinal  fluid  for  about 
two  days,  part  of  the  haemoglobin  is  converted  into  a  yellow 
pigment ;  if,  therefore,  after  centrifugalization  the  supernatant 
fluid  is  yellow,  it  indicates  that  the  blood  was  actually  present  in 
the  cerebro-spinal  fluid  whilst  in  the  body,  and  that  the  haemor- 
rhage had  occurred  two  days  or  more  previously.  If  the  super- 
natant fluid  is  colourless,  the  blood  came  from  the  puncture,  or 
had  only  been  recently  effused. 

If,  as  sometimes  happens,  you  can  only  get  a  few  drops  of  fluid 
from  a  lumbar  puncture,  it  is  not  wise  to  attach  any  importance 
to  the  presence  of  a  moderate  amount  of  blood.  The  needle 
always  picks  up  some  corpuscles  in  its  passage  through  the 
parietes,  and  these  may  be  quite  obvious  if  not  diluted  with  a 
considerable  amount  of  fluid. 

Large  endothelial  cells   containing   numerous  red   corpuscles 

16 — 2 


244  CLINICAL    BACTERIOLOGY   AND    HEMATOLOGY 

(similar  cells  to  those  shown  on  Plate  X.,  Fig.  3)  may  appear  in 
the  fluid  after  a  cerebral  haemorrhage.  They  do  not  make  their 
appearance  until  three  or  four  days  after  the  blood  has  been 
poured  out. 

H(smovrhage  into  the  meningeal  cavities  or  into  the  ventricles  is,  of 
course,  accompanied  by  blood  in  the  cerebro-spinal  fluid.  This 
may  be  found  in  cases  of  fracture  of  the  skull,  especially  of  the 
base,  or  of  the  spinal  column,  or  in  contusion  of  the  brain,  and  it 
is  worth  noting  that  in  some  cases  there  has  been  great  reUef  of 
the  symptoms  after  the  fluid  has  been  drawn  off".  Lumbar 
puncture  should  always  be  remembered  as  a  means  of  diagnosis 
in  patients  found  unconscious. 

"Septic"  Meningitis — i.e.^  that  due  to  the  meningococcus, 
pneumococcus,  streptococcus,  typhoid  bacillus,  etc. — is  character- 
ized by  the  presence  of  large  numbers  of  polynuclear  cells  in  the 
fluid  (see  p.  133).  In  all  cases  the  numbers  tend  to  be  more 
numerous  than  in  tuberculous  meningitis,  and  may  run  into 
hundreds  of  thousands  per  cubic  millimetre,  in  which  case  the 
fluid  resembles  watery  pus. 

The  importance  of  this  fact  is  very  great,  and  the  discovery  of 
polynuclear  leucocytoses  has  led  to  the  discovery  that  mild  cases 
of  this  affection  occur  which  are  hardly  diagnosable  clinically,  and 
which  may  be  completely  cured.  According  to  several  French 
authorities,  the  first  indication  of  commencing  cure  is  the  appear- 
ance of  lymphocytes,  which  must  therefore  be  looked  on  as  a 
good  sign  ;  after  a  time  the  polynuclears  become  less  and  less 
numerous,  and  finally  the  lymphocytes  alone  remain,  and  may 
persist  for  some  time  after  cure  has  taken  place.  I  have  watched 
several  septic  cases  recover,  and  have  seen  this  occur  two  or 
three  times.  The  appearance  of  lymphocytes  in  an  acute  septic 
meningitis  may  therefore  justify  a  good  prognosis. 


APPENDIX 


The  following  tables  may  be  useful  if  stains,  etc.,  have  to  be 
prepared  when  the  metric  weights  and  measures  are  not  at  hand. 
The  equivalents  given  are  not  absolutely  accurate,  but  are 
sufficiently  near  for  most  purposes : 

I  Litre     -         -         -         -         35I  fluid  ounces. 

100  c.c.    -         -         -         -  3!  fluid  ounces. 

I  Gramme       -         -         -  i5'43  grains. 

I  Fluid  ounce  -         -         -         28*4  c.c. 

I  Fluid  drachm        -         -  y^  c.c. 

I  Grain  -         -         -         -         0*064  gramme. 

To  convert  grammes  per  litre  into  grains  per  ounce,  multiply  by 
the  factor  ^^.  Thus,  10  grammes  per  litre  =  10  x  ^^^  =  4-4  grains 
per  ounce. 

To  convert  cubic  centimetres  per  litre  into  minims  per  ounce, 
multiply  by  the  factor  |f.  Thus,  25  c.c.  per  litre  =  25  x  ^f  =  12 
minims  per  ounce.    (A  close  approximation  is  given  by  multiplying 

To  convert  degrees  Centigrade  into  degrees  Fahrenheit,  multiply 
by  the  factor  |  and  add  32. 

II 

Neisser's  Method  of  Staining  the  Diphtheria  Bacillus. 

The  following  method  has  been  placed  in  the  Appendix,  as 
there  is  some  doubt  as  to  its  value.  It  is  a  method  by  which  the 
so-called  polar  bodies  are  stained  with  methylene  blue,  while  the 
rest  of  the  organism  is  coloured  a  faint  yellowish-brown  with 
Bismarck  brown. 

Cultures  should  be  made  on  blood-serum,  and  should  not  be  less 

24s 


246  CLINICAL    BACTERIOLOGY    AND    HEMATOLOGY 

than  nine  nor  more  than  twenty -four  hours  old.     Films  are  spread 
in  the  ordinary  way  and  stained  for  half  a  minute  in — 

Methylene  blue         .         .         .  i  gramme. 

Alcohol  (96  per  cent.)       -         -  20  c.c. 

Glacial  acetic  acid    -         -         -  50  c.c. 

Water      -----  g^o  c.c. 

They  are  then  washed  and  treated  for  half  a  minute  with — 

Bismarck  brown       .         -         .  5  grammes. 

Water      -----         1000  c.c. 

The  polar  bodies  are  small  spheres  which  are  contained  in  the 
bacilli,  there  being  usually  two  in  each  bacillus,  one  at  each  end. 
In  a  film  specimen  of  the  true  diphtheria  bacillus  stained  in  this 
way  they  appear  as  very  minute  dark  blue  or  black  dots,  which 
may  easily  be  mistaken  for  cocci ;  the  bodies  of  the  bacilli  are 
often  almost  invisible.  According  to  some  authorities,  the 
presence  of  these  granules  in  young  cultures  of  bacilli  which 
present  the  morphological  characters  of  the  diphtheria  bacillus  is 
proof  of  their  virulence,  whilst  their  absence  proves  the  cultures 
to  be  of  the  non-virulent  "pseudo-diphtheria"  bacillus.  The 
method  can  also  be  applied  to  films  made  directly  from  the  swabs, 
and  recent  researches  seem  to  prove  that  the  results  thus  obtained 
are  of  considerable  diagnostic  value. 

If  this  method  be  adopted  the  films  must  be  very  carefully 
searched,  as  it  often  happens  that  characteristically  stained  bacilli 
may  be  seen  in  one  part  of  the  field,  while  they  are  entirely  absent 
elsewhere.  This  is  especially  true  of  films  made  direct  from  the 
swabs. 

The  author  is  inclined  to  attach  very  considerable  importance 
to  a  positive  result  obtained  with  this  method  of  staining,  but 
would  not  consider  a  negative  result  as  indicating  the  absence  of 
the  diphtheria  bacillus. 

Ill 

Leishman's  Method  of  Staining  the  Spirochete  of 
Syphilis. 

I  have  seen  better  specimens  of  the  spirochaete  obtained  by 
this  method  than  by  any  other,  but  rny  own  results  have  been  less 
satisfactory..   Prepare  films  in  thp, usual  manner,  allow  them  to 


APPENDIX  247 

dry,  and  fix  by  means  of  methyl  alcohol  for  five  minutes ;  then 
pour  off  the  spirit  and  allow  the  films  to  dry  again.  Next  spread 
a  little  fresh  blood-serum  (obtained  from  a  Wright's  pipette  in  the 
usual  way)  in  a  thin  film  over  the  specimen,  using  the  end  ^f 
a  slide  as  a  spreader.  When  dry,  flood  the  slide  (or  cover-glass) 
with  Leishman's  stain  diluted  with  an  equal  volume  of  distilled 
water.     Allow  it  to  act  for  one  hour,  wash,  blot,  and  dry. 

IV 

Demonstration  of  Tubercle  Bacilli  in  Clots  from 
Pleuritic  Fluid,  etc — Improved  Method. 

This  method  yields  better  results  than  the  simple  digestion  of 
the  clot  previously  referred  to.  Remove  the  clot  from  the  fluid 
and  place  it  on  a  large  filter-paper  on  a  funnel,  so  that  the  fluid 
can  drain  away.  When  it  has  shrunk  to  a  small  size  remove  it 
and  place  it  in  a  large  volume  of  water  (not  necessarily  distilled), 
and  keep  in  gentle  movement  for  a  few  minutes.  Repeat  the 
process  two  or  three  times,  so  as  to  wash  away  all  haemoglobin, 
soluble  proteid,  etc.,  from  the  clot.  Place  the  latter  in  a  clean 
(preferably  new)  test-tube,  add  4  to  5  c.c.  of  water,  a  drop  of 
dilute  (i  to  10)  HCl,  and  a  pinch  of  pepsin,  shaking  until  the 
latter  is  dissolved.  Keep  it  in  the  incubator  until  the  clot  is  com- 
pletely dissolved,  adding  a  crystal  of  thymol  to  prevent  excessive 
bacterial  growth.  Then  shake  the  fluid,  put  it  in  a  clean  centri- 
fugalizing  tube,  and  centrifugalize  thoroughly.  Prepare  films 
from  the  exudate  and  stain  in  the  usual  way,  taking  great  care  to 
avoid  over-decolorization  j  one  or  two  dips  in  2J  per  cent,  sul- 
phuric acid  will  usually  suffice. 

The  clot  from  a  pint  or  more  of  fluid  can  be  treated  in  this 
way  ;  the  more  the  material  taken,  the  greater  the  chance  of 
success. 


OF 


P.  MUs  Sun  6. 
Mmm.  w    "' 


INDEX 


Abscess,  bacteriology  of,  102 

cold,  105 

collection  of  pus  from,  102 

leucocytosis  in,  223 

of  liver,  219 
Achorion  Schonleinii,  99 
Acid-fast  bacilli,  61 
Acid,  hydrochloric,  test  for,  116 

lactic,  test  for,  117 

stains,  203 
Actinomycosis,  65 
Adamson's  staining  method,  97 
Agar,  14 
Anaemia,  179,  191 

in  infancy,  221 

pernicious,  217 

secondary,  217 

splenic,  220 

Von  Jaksch's,  221 
Anaerobic  bacteria,  47 
Angina,  106 

scarlatinal,  109 

simple,  106 

syphilitic,  no 

Vincent's,  107 
Aniline  gentian  violet,  30 
Ankylostomiasis,  212 
Anthrax,  54 
Antitoxin,  diphtheria,  37 

tetanus,  45 
Antrum,  suppuration  of,  in 
Aortic  lesions,  blood  in,  231 
Appendicitis,  212,  229 
Arthritis,  bacteriology  of,  126 

gonorrhoeal,  84,  127 

tuberculous,  127 
Ascites,  cytology  of,  240 
Asthma,  blood  in,  230,  212 

sputum  in,  114 

Bacilluria,  121 
Bacillus,  acid-fast,  61 

of  anthrax,  54 
in  blood,  139 

of  Boas  and  Oppler,  115 


Bacillus,  "bottle,"  100 

coli,  69,  105 

in  urine,  119 

comma,  91 

of  diphtheria,  40,  42 

Ducrey's,  94 

fusiformis,  108 

geniculatus,  115 

of  glanders,  68,  105 

Hoffmann's,  44 

of  influenza,  52 

of  Koch  and  Weeks,  112 

of  leprosy,  65 

mallei,  68,  105 

of  Morax  and  Axenfeld,  112 

Pfeiffer's,  52 

of  plague,  93 

pyocyaneus,  102,  104,  105 

of  smegma,  61 

of  soft  sore,  94 

of  tetanus,  47 

of  tubercle,  60.  See  also  Tubercle 

of  typhoid  fever,  69,  105 
in  blood,  69 
in  urine,  119 

vaginae,  88 

xerosis,  113 
Bacteriuria,  121 
Basic  stains,  203 
Basophile  cells,  208 
Bilharzia  disease,  blood  in,  212 
Bismarck  brown,  246 
Blood,  bacteriology  of,  138 

collection  of,  for  serum,  55 

crisis,  215 

cultures,  69,  138 

-films,  fixation  of,  202 
preparation  of,  198 
staining  of,  203 

opsonic  index  of,  148 

platelets,  209 

-serum  culture  medium,  15 
Boils,  104 

Borax  methylene  blue,  29 
Bottle  bacillus,  100 


248 


INDEX  249 


Bottles,  sterilization  of,  7 
Bouillon.     See  Broth 
Bronchitis,  230 
Broncho-pneumonia,  230 
Broth,  preparation  of,  10 
Bubo  in  plague,  93 
in  soft  sore,  95 
Bulloch,  154 

Capsules  of  pneumococci,  51 

Wright's  blood,  33 
Carbol  fuchsin,  29 

gentian  violet,  30 

thionin,  30 
Carcinoma  ventriculi,  blood  in,  228 
Catarrhalis,  micrococcus,  53,  54 
Cathcart's  microtome,  163 
Cells,  alveolar,  114 

bronchial,  114 

endothelial,  236 

malignant,  237 

squamous,  114 
Cerebral   tumour,   lumbar   puncture 
in,  242 
cerebro-spinal  fluid  in,  242 
Cerebro-spinal  fluid,  132 

cytology  of,  133,  241 

fever,  134.  135 
Cleaning  blood  pipettes,  195 

cover-glasses  and  slides,  31 
Chlorosis,  218 
Cholera,  91 

Clumping  of  bacteria,  71 
Cirrhosis,  blood  in,  229 
Colour-index,  192 
Comma  bacillus,  91 
Conjunctiva,  bacteriology  of,  11 1 
Cornet's  forceps,  23 
Cornil's  myelocytes,  207 
Corpuscles,  red,  enumeration  of,  181 

morphology  of ,  213 
Cotton-wool,  sterilization  of,  8 
Cover-glasses,  cleaning,  31 
Crescents  in  malaria,  143 
Cultures  from  blood,  143 

from  cerebro-spinal  fluid,  137 

from  pus,  103 

from  throat,  106 

examination  of,  22 

incubation  of,  18 

inoculation  of,  16 

media,  9 

stab  and  stroke,  13 

sterilization  of,  8 
Cysticercus,  eosinophilia  in,  212 
Cystitis,  bacteriology  of,  119 
Cyto-diagnosis,  233 

Degeneration,  glycogenic,  210 
of  leucocytes,  235 
of  red  corpuscles,  214 


Dermatitis   herpetiformis,    blood   in 

232 
Dewar's  flask,  21 
Differential  leucocyte  count,  209 
Diphtheria,  37 
Diplococcus  meningitidis  intracell«-- 

laris,  136 
Ducrey's  bacillus,  94 
Durham,  71 

Ehrlich's  myelocytes,  208 

triacid  stain,  204 
Embedding  in  paraffin,  160,  166 
Empyema,  124,  230 
Endocarditis,  blood  in,  230 

ulcerative,  50 
Endothelial  cells,  236 
Enteric  fever.     See  Typhoid  fever 
Enumeration  of  bacteria  in  water,  12 
in  vaccines,  156 

of  leucocytes,  193 

of  red  corpuscles,  181 
Eosin,  30,  203 
Eosinophile  leucocytes,  208,  211 

myelocytes,  208,  213 
Eosinophilia,  211 

Epilepsy,  cerebro-spinal  fluid  in,  242 
Erysipelas,  50 
Erythema  multiforme,  232 
Ewing,  225 

Faeces,  tubercle  bacilli  in,  63 
Fauces,  bacteriology  of,  105 

cells  from,  114 
Favus,  99 
Fever,  puerperal,  223,  231 

relapsing,  140 

scarlet,  226 
Films,  blood,  fixation  of,  202 
preparation  of,  198 
staining  of,  203 

of  exudates,  234 

of  sputum,  62 
Fixation  of  blood-films,  202 

of  tissues,  160,  162 
Flasks,  sterilization  of,  7 
Follicular  tonsillitis,  106 
Folliculitis,  104 
Foulerton,  88 

Fragmentation  of  nucleus,  235 
Freezing  process,  160,  163 
Fuchsin,  carbol,  29 

Gastric  contents,  examination  of,  115 
Gelatin,  13 

liquefaction  of,  12,  58 
General  paralysis,  cerebro-spinal  fluid 

in,  241 
Gentian  violet,  aniline,  30 
German  measles,  227 
Giemsa's  stain,  90 


250 


INDEX 


Glanders,  68 
Glycogen  reaction,  210 
Gonococcus,  84 

in  blood,  140,  145 

in  conjunctiva,  112 

in  joints,  127 

in  meninges,  134 

in  urine,  120 
Gowers'  haemoglobinometer,  175 
Gram's  iodine,  30 

method,  24,  172 
Granular  degeneration,  215 
Griinbaum,  71 

Haematoxylin,  staining  by,  165.  171 
Haemocytometer,  Thoma's,  182 
Haemoglobin,  estimation  of,  175 
Haemoglobinometer,  Gowers',  175 

Haldane's,  176 

Oliver's,  177 
Haemorrhage  into  meninges,  243 
Hairs,  ringv^^orm  in,  95 
Haldane's  haemoglobinometer,  176 
Hanging-drop  preparation,  73 
Hanot,  229 

Herpes,  cerebro-spinal  fluid  in,  241 
Herxheimer,  90 
Hevi^lett,  41 
Hodgkin's  disease,  220 
Hoffmann's  bacillus,  44 
Hooping-cough,  227 
Horder,  228 

Hyaline  leucocytes,  large,  207 
Hydatid  disease,  blood  in,  212 
Hydrochloric  acid,  test  for,  116 
Hysteria,  cerebro-spinal  fluid  in,  242 

Impetigo,  104 
Incubation  of  cultures,  18 
Incubators,  19 
Infancy,  anaemia  in,  221 
Influenza,  52 

blood  in,  227 
Inoculation  of  cultures,  16 
Iodine  reaction,  210 

Gram's,  30 

Jenner's  stain,  204 
Joints,  fluids  from,  126 
Justus's  test,  180 

Kerion,  99 

Koch-Weeks  bacillus,  112 

Kiihnau,  147 

Lactic  acid,  test  for,  117 
Large  hyaline  leucocytes,  207 
Lead-poisoning,  blood  in,  215 
Leishman's  stain,  205 
Leprosy,  65 
Leptothrix,  no 


Leucocytes,  enumeration  of,  in  blood, 

193 
in  cerebro-spinal  fluid,  241 

morphology  of,  206 
Leucocythaemia,  219 
Leucocytosis,  196 
Leucopenia,  198 

Liquefaction  of  gelatin,  12,  58,  104 
Liver,  abscess  of,  229 

cirrhosis  of,  229 

examination  of,  158 

hydatid,  blood  in,  229 
Locomotor  ataxy.     See  Tabes 
Loffler's  blue,  29 
Lumbar  puncture,  128 
Lupus,  opsonic  index  in,  154 
Lymphocytes,  206,  210 
Lymphocytosis,  210 

of  cerebro- spinal  fluid,  210,  242 
Lymphosarcoma,  220 

Macrocytes,  213 
Malaria,  blood  in,  226 

parasites  of,  142 
Malignant  disease,  blood  in,  228 

exudates,  cells  of,  237,  239 

pustule,  54 
Manson's  methylene  blue,  29 
Mast  cells,  208,  213 
McCrae,  228 
Measles,  226 

German,  227 
Media,  culture,  preparation  of,  g 
Megaloblasts,  215 
Megalocytes,  213 
Megalosporon  ectothrix,  99 

endothrix,  98 
Meningitis,  132,  134 

tuberculous,  133,  137,  227 
Meningococcus,  136 
Methylene  blue,  29 
acid,  30 
Loffler's,  29 
Manson's,  29 
Micrococcus  catarrhalis,  53,  106 

epidermidis,  26 

ureae,  120 
Microcytes,  213 
Microscope,  choice  of,  i 

use  of,  27 
Microsporon  Audouini,  97 

furfur,  loi 
Microtomes,  163,  168 
Milk,  tubercle  bacilli  in,  63 
Mitral  lesions,  blood  in,  230 
Mononuclear  leucocytes,  large,  207 
Morax-Axenfeld  bacillus,  112 
Morbus  caeruleus,  blood  in,  231 
Mouth,  bacteriology  of,  105 
Mycelium,  67 
Myelocytes,  207,  211 


INDEX 


251 


Myelocytes,  eosinophile,  208,  213^ 
Myelogenous  leucocythaemia,  219 

Nails,  ringworm  of,  100 
Needles,  platinum,  16 
Negative  staining,  51 
Neisser's  method,  245 

stain,  245 
Nile  blue,  90 
Normoblasts,  215 
Nose,  bacteriology  of,  no 
Nutrient  gelatin,  11 

Oidium  albicans,  109 

Oil-immersion  lens,  28 

Oliver's  haemoglobinometer,  177 

Opsonic  index,  148 

Osier,  228 

Osteomyelitis,  105 

Otitis  media,  50 

Ovarian  tumours,  blood  in,  232 

Paraffin  process,  166 

Parametritis,  231 

Parasites,  animal,  211 

Pemphigus,  212,  232 

Perimetritis,  231 

Peritoneal  fluid,  cytology  of,  240 

Peritonitis,  50 

blood  in,  213,  229 

tuberculous,  240 
Petri  dishes,  sterilization  of,  8 
Pfeiffer's  bacillus.     See  Influenza 
Pipettes,  32 

Wright's,  151 
Plague,  93 
Platelets,  blood,  209 
Plating,  isolation  of  bacteria  by,  12, 

51,  103 
Platinum  needles,  16 
Pleurisy,  50,  123 

blood  in,  229 

cytology  of,  237 

due  to  infarcts,  240 

malignant,  238 

rheumatic,  238 

septic,  238 

tuberculous,  123,  237 
Pneumococcus,  arthritis  due  to,  126 

in  blood,  139 

capsules  of,  51 

characters  of,  51 

cultivation  of,  126 

empyema  due  to,  124 

lesions  due  to,  49 

meningitis,  134 
Pneumonia,  blood  in,  225 

lobar,  bacteriology  of,  49 

lobular,  bacteriology  of,  49 

opsonic  index  in,  154 
Poikilocytes,  214 


Polar  bodies,  246 

staining,  93 
Polychromatophil  degeneration,  214 
Polynuclear  leucocytes,  207,  210 
Post-mortem  examinations,  158 

Potato  culture  medium,  15  ^     

Proteus  vulgaris,  119 
Pseudo-leukaemia,  230 
Psoriasis,  loi 

Puerperal  fever,  blood  in,  231 
Pulmonary  anthrax,  54 
Purpura  haemorrhagica,  228 
Pus,  actinomycotic,  66 

bacteriology  of,  102 

collection  of,  102 

pleuritic,  124 

pneumococcic,  50 

tuberculous,  63 
Pustule,  malignant,  54 
Pyelitis,  119 
Pyosalpinx,  231 

Ray  fungus,  66 

Red  corpuscles,  enumeration  of,  181 

morphology  of,  213 
Relapsing  fever,  140 
Rheumatic  pleurisy,  238 
Rheumatism,  227 
Ringworm,  95 

staining  of,  96,  97 
Rocking  microtome,  168 

Sabouraud, 99 

Saccharomyces  albicans,  109 
Sarcinae,  117 
Scarlatinal  angina,  109 
Scarlet  fever,  blood  in,  226 
Schaudinn,  89 
Seborrhoea,  loi 
Section  cutting,  159 

staining,  165,  170 
Sepsis,  210,  222 
Septicaemia,  50 

Serous  cavities,  fluids  from,  121 
Skin    diseases,    eosinophilia  in,  212, 

232 
Slides,  cleaning,  31 
Smegma  bacillus,  61 
Soft  sore,  94 
Spirillum  of  relapsing  fever,  140 

Vincent's,  109 
Spirochaeta  pallida,  89 

refringens,  89 
Spleen,  examination  of,  158 
Splenic  anaemia,  220 
Spleno-medullary  leucocythaemia,  219 
Spores,  II,  48,  55 
Sputum,  collection  of,  61,  113 

cytology  of,  114 

in  influenza,  53 

pneumococci,  51 


252 


INDEX 


Sputum,  tuberculous,  60 
Stab  and  stroke  cultures,  13 
Staining,  negative,  51 
Stains,  28 

acid,  203 

basic,  203 

Ehrlich's,  204 

Jenner's,  205 

Leishman's,  205 

triacid,  204 
Staphylococci,  104 

in  blood,  139,  148 

in  joints,  127 

in  meningitis.  134 

in  pleura,  125 

in  urine,  120 
Sterilization,  5 

of  cultures,  8 

by  dry  heat,  6 

intermittent,  11 

of  skin,  122,  130 

by  steam,  8 
Stomach,  carcinoma  of,  115,  228 

dilatation  of,  117 
Streptococci,  104 

in  blood,  139,  148 

in  joints,  127 

in  meningitis,  134 

in  pleura,  124 

in  urine,  120 
Streptothricosis,  65 
Suppuration.     See  Pus 
Swabs,  38 
Syphilis,  89 

blood  in,  228 

cerebro-spinal  fluid  in,  241 

Justus's  test,  180 
Syringomyelia,    cerebro-spinal    fluid 
in,  242 

Tabes,  cerebro-spinal  fluid  in,  241 
Test-tubes,  sterilization  of,  7,  8 
Tetanus,  47 
Thionin,  30 
Throat  swabs,  38 
Thrush,  log 
Tinea  versicolor,  loi 
Toison's  fluid,  183 
Tonsillitis,  106 
Treponema  pallidum,  89 
Triacid  stain,  204 
Trichinosis,  212 
Trichophyton  ectothrix,  99 
endothrix,  98 


Tubal  gestation,  blood  in,  232 
Tubercle,  bacillus  of,  60 
■   in  blood,  139 

in  conjunctiva,  113 

in  joints,  127 

in  meninges,  134,  137,  241 

in  pleura,  125 

in  pus,  105 

in  sputum,  60 
Tuberculin,  64,  157 
Tuberculosis,  blood  in,  227 

opsonic  index  in,  154 
Tuberculous  meningitis,  134,  137,242 

pleurisy,  125,  237 
Tumours,  malignant,  blood  in,  228 
Turk,  227 
Typhoid  fever,  69 

blood  in,  213 

Widal's  reaction,  70,  72,  77 

Ulcer  of  stomach,  229 
Ulcerative  endocarditis,  50,  104 
Unna's  bacillus,  94 

bottle  bacillus,  100 
Uraemia,  134 
Urine,  bacteriology  of,  118 

gonococci  in,  87 

tubercle  bacilli  in,  62 
Urticaria,  212 

Vaccines,  104,  155 
Vagmae,  bacillus,  88 
Vaginitis,  88 
Valvular  disease,  230 
Vibrio  of  cholera,  91 
Vincent's  angina,  107 
Vomit,  examination  of,  115 
Von  Jaksch's  anaemia,  221 

Water,  examination  of,  12 

Weichselbaum,  136 

Weights  and  measures,  245 

Whitfield.  loi 

Whooping     cough.       See     Hooping 

cough 
Widal's  reaction,  70,  72,  77 
Wool-sorter's  disease,  54 
Wright's  blood  capsules,  33 
opsonic  method,  149 
pipettes,  151 

Xerosis  bacillus,  113 

Yeasts,  116 


H.  K.  Lewis,  136,  Gower  Street,  London.  IV.C. 


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Clinical  "bactt 


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